Support and packaging for membranes

ABSTRACT

A support assembly for supporting a biological product (e.g., membrane) in an operative position. The support assembly has a base and a cover. A membrane receiving portion of the base defines a plurality of perforations that extend between top and bottom surfaces of the product receiving portion. The cover is releasably coupled to the base in a product-covering position in which the cover overlies the product receiving portion of the base. In the operative position, the biological product engages the top surface of the product receiving portion and the bottom surface of the cover.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/657,535, filed Mar. 13, 2015, now U.S. Pat. No. 10,279,974, whichclaims priority to and the benefit of the filing date of U.S.Provisional Patent Application No. 61/953,716, filed Mar. 14, 2014. Eachof the above-identified applications is hereby incorporated herein byreference in its entirety.

BACKGROUND

Products are increasingly being used for treatment of wounds, burns,lacerations or surgical excisions. However, to use such products, thereneeds to be a method of manufacturing, packaging and applying theproduct that maintains the integrity of the product (includingmembranes) during these processes. Conventional packaging for applying abiological or membrane product does not lend itself to convenientapplication, and had multiple failings including its inability toprovide real time sizing and directionality, among others. Conventionalbandages and dressings, for example, fail to adequately protectlarge-scale, deep, oddly shaped and other types of wounds or tissuedefects. Therefore, various alternatives have been explored in the art.Among these alternatives are split- and full-thickness grafts of cadaveror porcine skin, human allografts, cultured skin equivalents andautografts. Most of these membranes, including tissue or tissueequivalent products/synthetic products contain, in at least someaspects, a morphology similar to actual human skin, which has anepithelial layer on the top and connective tissue with fibroblasts orother types of cells on the bottom facing the wound and/or damagedtissue. Such products can be considered to have directionality. Further,when such membranes are used to treat a variety of wounds (or tissuedefects), the preferred orientation of the wound, tissue, graft orapplied biological product is such that the connective tissue layerrests on the wound bed while the epithelial layer is away from the woundbed.

Challenges exist with conventional packaging systems for the storage,transport and the delivery or application of membranes to various humanor animal structures needing treatment such as wounds or tissue defects.For example, the tensile strength of the grafts, tissues, or membranesis such that they often cannot support their own weight and tear ifsuspended by an edge. For this reason, these types of graft, tissue ormembrane products are often mounted on a carrier paper and then packagedinto a sealable container (such as a bag), which contains a substantialamount of liquid (e.g., a biomedium such as a biosolution orbioprotectant). Typically, the attachment of the graft to the carrierpaper, however, is relatively weak. Thus, during manufacture,transportation to its end use site, and finally during handling prior toapplication to a wound (or tissue defect), the tissue or membrane mayseparate from the carrier paper voluntarily or inadvertently, due toshear forces of liquid moving around in the overall packaging. As aresult, the tissue, graft or membrane to be applied may curl, attach toitself, attach to other aspects of the packaging, tear, or in some otherfashion become unusable for final application to the human or animal.This results in significant waste, time loss, patient and/or careprovider dissatisfaction and cost, and ineffective therapeutic treatmentof the wound or tissue defect, among other negative attributes. Inaddition, if a graft or membrane product being supplied iscryopreserved, complete thawing of all ice crystals (e.g., of thebiomedium or cryoprotectant contained in the container along with thetissue, graft, or membrane to be finally applied) is necessary prior tothe product's final application to a human or animal. This thawingprocedure can last for several minutes (e.g., up to 30 minutes or more)depending upon the volume of liquid and other material to be thawedwithin the packaging. This thawing wait time and additional proceduremake such conventional tissue, graft or membrane products and productpackaging inconvenient for health care providers who may be treatingseveral wounds during any given period of time.

Finally, concerns also exist with current conventional application anddelivery of tissue, graft or membrane-based products/systems/packages.If the membrane, tissue or graft needs to be separated from thepackaging (e.g., a carrier paper or carrier bottom paper) and at thesame time kept in a proper orientation (e.g., epithelial on top andconnective tissue on the bottom) for delivery to the patient site suchas a wound (or tissue defect), then the packaging must so indicate in aclear manner and be capable of maintaining that orientation duringmanufacture, transit and final application This becomes even moredifficult to achieve when the size of the supplied graft, tissue ormembrane is small. Once the graft, tissue or membrane folds over uponitself (or becomes disorientated in some other fashion), it is verydifficult to restore the biological material to its original planarconfiguration, for example, and essentially impossible to make theappropriate final application to the wound.

Therefore, there is a need within the art for a new package, packagingsystem, composition, device, article of manufacture and method ofdelivery utilizing such materials that overcomes these deficiencieswithin the conventional art.

SUMMARY

Described herein, in one aspect, is a support assembly for supporting abiological product (e.g., a membrane) in an operative position. Thesupport assembly can have a base and a cover. The base can have alongitudinal axis and comprise a product receiving portion. The productreceiving portion can have a top surface and an opposed bottom surfacethat are spaced apart relative to a vertical axis that is perpendicularto the longitudinal axis of the base. The product receiving portion canhave at least one traction-creating feature, which can be selected fromthe group consisting of (i) a rough top surface; and (ii) a plurality ofperforations that extend between the top and bottom surfaces of theproduct receiving portion. The cover can have a longitudinal axis, a topsurface, and an opposed bottom surface. The cover can be configured forreleasable coupling (optionally, attachment) to the base in aproduct-covering position. In the product-covering position, the coveroverlies the product receiving portion of the base. The base and thecover can be configured to cooperate to support the biological productin the operative position. In the operative position, the biologicalproduct is positioned in engagement with at least a portion of the topsurface of the product receiving portion of the base and at least aportion of the bottom surface of the cover.

In another aspect, described herein is a membrane product packagecomprising a membrane and a support assembly as disclosed herein. Themembrane product package includes a membrane that is positioned in anoperative position between the product receiving portion of the base andthe cover. The membrane can be positioned in engagement with at least aportion of the top surface of the product receiving portion of the baseand at least a portion of the bottom surface of the cover.

Also described is a method of producing a membrane product package asdisclosed herein. The method can comprise positioning a membrane in anoperative position between the product receiving portion of the base andthe cover of a support assembly as disclosed herein. The membrane can bepositioned in engagement with at least a portion of the top surface ofthe product receiving portion of the base and at least a portion of thebottom surface of the cover.

Additionally, described herein is a method of applying a membrane usinga membrane product package as disclosed herein. The method can compriseremoving the cover from the membrane product package to expose a topsurface of the membrane. Following removal of the cover, the membranecan remain in the operative position, which generally corresponds to theorientation in which the membrane is to be applied. The method canfurther comprise sliding the membrane relative to the top surface of theproduct receiving portion of the base to disengage the membrane from thetop surface of the product receiving portion of the base and permitselective application of the membrane as further disclosed herein.

Further described is a kit for repairing a tissue defect. The kit cancomprise a membrane product package as disclosed herein and instructionsfor applying the membrane to repair the tissue defect.

More generally, in some aspects and embodiments, the present technologyprovides a device (e.g., a support assembly), composition (e.g., amembrane product package), article of manufacture or system comprising:a base comprising at least one product (e.g., membrane) receivingportion; a cover; and at least one location in which the base and thecover are in communication (e.g., coupled to one another through amembrane or attached to one another as further disclosed herein). Insome embodiments, the device, composition, article of manufacture orsystem further comprises a membrane, tissue, graft or other biologicalmaterial(s) temporarily connected, attached, adhered, or operativelyassociated with the cover, the base or both. The base and the cover canbe positioned in communication via at least one temporary or removableattachment between the base and the membrane, the membrane and thecover, and/or the base and the cover.

In some aspects or embodiments, the product (e.g., membrane) receivingportion of the present technology comprises a structured surface that isconfigured to promote adhesion of a membrane or other biological productto the product receiving portion as further disclosed herein. Suchstructured surfaces are referred to herein as “traction-creatingfeatures.” In other aspects or embodiments of the present technology,the traction-creating features can include one or more of a roughsurface; a plurality of perforations; a surface comprising a pluralityof channels; a surface comprising a plurality of grooves; a surfacecomprising a plurality of indentations; or a surface comprising aplurality of porations. In exemplary aspects, the rough surface can beone or more of an abraded surface, a scratched surface, an unevensurface, a gritty-type surface (yet, preferably free or substantiallyfree of loose particulate), or a bumpy surface, among others. In someaspects, the traction-creating feature comprises at least oneperforation, at least one channel, at least one groove or at least oneindentation, wherein, in some instances, the at least one perforation,at least one channel, at least one groove and/or at least oneindentation has a complex pattern. In some aspects or embodiments, thebase further comprises a handling portion, which can optionally beadjacent to the membrane receiving portion. In further aspects orembodiments, the handling portion does not overlap with the cover. Instill further aspects or embodiments, the handling portion comprises atleast one tab. In some aspects or embodiments, the tab spans the entirewidth of the base. In other aspects or embodiments, the cover spans theentire product (e.g., membrane) receiving portion of the base. However,segments, portions or parts of the width of the base or membranereceiving portion are also envisaged.

In additional aspects or embodiments, the at least one location in whichthe base and cover are in communication (preferably temporarily) witheach other comprises, for example, at least one cauterization point, atleast one point made by an ultrasonic welder, or at least one pointcomprising a biocompatible adhesive. In other aspects or embodiments,the at least one location in which the base and cover are incommunication (preferably temporary communication) with one anothercomprises a plurality of points. In still further aspects andembodiments of the present technology, the base and the cover are formedfrom a single piece of biocompatible plastic or other suitablebiopolymer suitable for use with membranes, tissues, grafts, or otherbiological materials. In additional aspects or embodiments of thepresent technology, the base and the cover are separate pieces ofbiocompatible plastic or biopolymer or other biocompatible material. Inother aspects or embodiments, the base and the cover are made of thesame type of biocompatible plastic or other biopolymer, biocopolymer orother biocompatible material.

Some aspects of the present technology provide a composition comprising:a base comprising at least one membrane receiving portion; at least onemembrane; at least one cover; and at least one location in which thebase and the cover are in communication, wherein the membrane ispositioned between the base and the cover. In other aspects orembodiments, the base further comprises at least one handling portion.In still further aspects or embodiments, the handling portion can beadjacent to the membrane receiving portion.

Moreover, additional aspects or embodiments of the present technologyprovide a cryopreserved membrane composition comprising: a) any of thecompositions, devices, articles of manufacture, devices or systems ofthe present technology disclosed herein; and b) at least onecryopreservation medium or other compatible biological medium.

In other instances of the present technology a kit is providedcomprising: any of the devices, articles of manufacture, compositions,or systems of the present technology described herein; and instructionsor guides for sizing, orienting, and/or applying, connecting or adheringat least one membrane between the base and the cover of the device,wherein the base and the cover have at least one location which isadapted to be in communication with each other. Alternatively, theseaspects and embodiments of the present technology can also include atleast one, preferably more than one, point of connection between thecover and the membrane, the base and the membrane, and/or the cover andthe base. Additionally, the kit aspects and embodiments of the presenttechnology can further comprise an adhesive. The adhesive may be anadhesive that is biologically compatible, or other suitablebiocompatible materials to connect the cover and the base, to connectthe base to the membrane, or to connect the membrane-covered base to thecover. The adhesive may be biocompatible, able to withstand physical orchemical alterations by solutions and solvents (e.g., a cryopreservationsolution), and/or to withstand a wide range of temperatures (forexample, from about 60° C.±5° C. to about −196° C.±5° C., as describedherein.

In some aspects or embodiments of the present technology pertaining to akit, instructions can further comprise at least one method oftemporarily adhering, connecting, or applying the base to a first sideof the membrane, wherein the method comprises applying at least onebiocompatible adhesive to at least one location between the base and afirst side of the membrane to form a temporary bond between the membraneand the base. In some instances, the instructions further comprisemethods of applying the at least one adhesive to at least one locationon a first side of a cover and the second side of the membrane totemporarily bond the cover to the second side of the membrane, forming acover-membrane-base configuration.

In other aspects, the instructions included with the kits of the presenttechnology provide a method of temporarily and sufficiently coupling(e.g., connecting, attaching, applying, adhering, or indirectly securingthrough the membrane) the cover to the base wherein the membrane islocated between the cover and base (e.g., similar to a sandwich-likeconfiguration), wherein the method further comprises applying at leastone adhesive (or other biocompatible material) to at least one pointbetween the cover and the membrane-covered base and/or between the coverand the membrane.

It should be appreciated by those skilled in the art that otherattachment mechanisms and methods can be utilized to attach the cover tothe base as well as the cover to the membrane and to attach the base tothe membrane as well as the cover and base to the membrane. For example,in some aspects and embodiments of the present technology, the kitincludes instructions for cauterizing at least one point of the cover tothe base, wherein the membrane is located between the cover and base. Insome instances, the instructions provide a method of cauterizing themembrane to the base at least at one point, alternatively at least attwo points, alternatively at least at three points, alternatively atleast at four points, alternatively at least at five points,alternatively at least at six points. In other instances, theinstructions further provide instructions on cauterizing the cover tothe membrane-base at least at one point, alternatively at two points,alternatively at least at three points. The instructions provide amethod of cauterizing the cover, membrane and base such that themembrane is disposed between the cover and base. In alternative aspectsor embodiments of the present technology, the instructions can furthercomprise at least one method of maintaining the directionality of themembrane, the method comprising the step of adhering a first side of themembrane to the base in a specific orientation and/or direction desired(e.g., in the operative position).

In still further aspects or embodiments of the present invention, thekit can also further comprise at least one set of instructions forcryopreserving the device, composition, article of manufacture or systemof the present technology comprising at least one membrane to becryopreserved. With respect to these particular aspects and embodiments,the cryopreservation step comprises, for example, cryofreezing thedevice, composition, article of manufacture, or system of the presenttechnology containing the membrane at about −18 to −20° C.±5° C. toabout −196° C.±5° C., in some aspects from about −80° C. to about −196°C.±5° C. For acellular membranes, freezing may take place from about−18-−20° C.±5° C. to about −196° C.±5° C. For membranes containingviable cells, freezing may take place from about −45° C.±5° C. to about−196° C.±5° C. In aspects or embodiments of the present technology, thekit can further comprise instructions for thawing the cryopreservedmembrane while a component of the device, composition, article ofmanufacture, or system described herein.

In some aspects, the kit further comprises instructions for applying themembrane to a human or animal in need thereof.

In some instances, the present technology provides a kit for repairing atissue defect comprising: a cryopreserved composition, device, articleof manufacture, or system described herein; and instructions forapplying the cryopreserved membrane or biological material to the tissuedefect. In some aspects, the kit further comprises instructions forthawing the cryopreserved composition. In some aspects, the kitcomprises further instructions on maintaining the directionality of themembrane while being applied to the tissue defect. In some aspects, thekit further comprises instructions for removal of the cover. In someaspects, the kit further comprises instructions for maintaining thedirectionality of the membrane. In some aspects, the kit furthercomprises instructions for removing the membrane from the base.

In further instances, the present technology provides a method ofmaintaining the directionality of a membrane during storage,cryopreservation, or during application to a subject comprising:preparing a membrane, wherein the membrane is orientated having a firstand a second side (e.g., a top surface and a bottom surface), whereinthe first and second side comprise different compositions, structures orproperties; b) adhering the membrane to the device, system or article ofmanufacture described herein comprising a base and a cover, wherein themembrane is disposed between the base and the cover; and wherein thefirst side of the membrane is facing the base and the second side of themembrane is facing the cover; and wherein device further comprises alabel to indicate orientation.

In still other aspects, described herein is a method of applying amembrane to a human or animal in need thereof, comprising: obtaining acomposition, system or article of manufacture as described herein whichhas been cryopreserved and frozen; thawing the composition, system orarticle of manufacture; optionally rinsing the membrane in a sterilephysiological solution; removing the cover from the membrane and base;and applying the membrane from the base onto the human or animal toretain directionality of the membrane.

In yet further instances, the present technology provides a method ofmaintaining integrity of a membrane during cryopreservation, comprising:providing a device as described herein; adhering a membrane to at leastan area of the membrane receiving portion of the base; adhering thecover to the base (optionally, through the membrane, which can bedirectly adhered to the base), wherein the membrane is between the coverand the base; and placing the device comprising the membrane into acontainer; and contacting the container with sterile cryopreservationsolution, wherein the device comprising the membrane is submerged in thecryopreservation solution; and cryopreserving the container at atemperature of about −80° C. to about −196° C., wherein the integrity ofthe membrane is maintained once the membrane is thawed to roomtemperature.

In some aspects, described is a method of treating a wound comprisingapplying a membrane of any one of the compositions, systems or articlesof manufacture described herein to a human or animal in need thereof.

Some aspects provide a system comprising: a base comprising a membranereceiving portion; and a cover; and at least one location in which thebase and the cover are in communication. In some aspects, the systemfurther comprises a membrane, wherein the membrane is disposed betweenthe cover and the base. In these aspects, the base and the cover can bein communication at least one attachment point (at least one temporaryor removable attachment between the base and the cover, the base and themembrane, and/or the cover and the membrane).

In some aspects, an article of manufacture comprising: a base comprisinga membrane receiving portion; and a cover; and at least one location inwhich the base and the cover are in communication (at least onetemporary or removable attachment between the base and the cover, thebase and the membrane, and/or the cover and the membrane).

The present technology will be described in more detail below withregard to the devices, compositions, articles of manufacture, devices,systems and methods of utilizing each for the protection of tissues,membranes or graft materials, for example, during manufacture,processing, cryopreservation, storage, and transport.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict the base and the cover of an exemplary supportassembly as disclosed herein. FIG. 1A is a top view depicting the baseof the support assembly, and FIG. 1B is a top view depicting the coverof the support assembly.

FIGS. 2A and 2B depict the base and the cover of another exemplarysupport assembly as disclosed herein. FIG. 2A is a top view depictingthe base of the support assembly, and FIG. 2B is a top view depictingthe cover of the support assembly.

FIGS. 3A and 3B depict the base and the cover of another exemplarysupport assembly as disclosed herein. FIG. 3A is a top view depictingthe base of the support assembly, and FIG. 3B is a top view depictingthe cover of the support assembly.

FIGS. 4A and 4B depict the base and the cover of another exemplarysupport assembly as disclosed herein. FIG. 4A is a top view depictingthe base of the support assembly, and FIG. 4B is a top view depictingthe cover of the support assembly.

FIGS. 5A-5C schematically depict the assembly of a membrane productpackage as disclosed herein. FIG. 5A is an exploded view of the membraneproduct package, showing the relative orientation of the base, themembrane, and the cover. FIG. 5B is a top view of the base, showing themembrane positioned in engagement with the product receiving portion ofthe base. FIG. 5C is a top view of the membrane product packagefollowing positioning of the cover over the membrane, thereby supportingthe membrane between the cover and the product receiving portion of thebase. As shown, the membrane can be attached to the base and the coverat a plurality of attachment points as disclosed herein.

FIGS. 6A-6B depict an exemplary configuration of attachment points on anexemplary support assembly as disclosed herein. FIG. 6A is a top viewdepicting the attachment points on the base of the support assembly, andFIG. 6B is a top view depicting the attachment points on the cover ofthe support assembly.

FIGS. 7A-7B depict an exemplary configuration of attachment points onanother exemplary support assembly as disclosed herein. FIG. 7A is a topview depicting the attachment points on the base of the supportassembly, and FIG. 7B is a top view depicting the attachment points onthe cover of the support assembly.

FIG. 8 is an isolated top view of a plurality of perforations of theproduct receiving portion of a base, as disclosed herein.

FIG. 9 is an isolated top view of a product receiving portion having arough surface as disclosed herein.

FIG. 10 is a schematic representation of the perforation size andcenter-to-center spacing between perforations on amniotic membranehandling properties.

FIG. 11 is a schematic representation of the perforation pattern on theability to maintain more than 75% membrane on the base after removal ofthe cover.

FIG. 12 is a schematic representation of the cautery patterns foramniotic membranes.

FIG. 13 is a schematic representation of the cautery patterns forchorionic membranes.

DETAILED DESCRIPTION

The present invention now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, this invention may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout. It is tobe understood that this invention is not limited to the particularmethodology and protocols described, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention.

Many modifications and other embodiments of the invention set forthherein will come to mind to one skilled in the art to which theinvention pertains having the benefit of the teachings presented in theforegoing description and the associated drawings. Therefore, it is tobe understood that the invention is not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

As used herein the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cover” can include a plurality of such covers,and so forth. All technical and scientific terms used herein have thesame meaning as commonly understood to one of ordinary skill in the artto which this invention belongs unless clearly indicated otherwise.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

As used herein, the terms “optional” or “optionally” mean that thesubsequently described event or circumstance may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

As used herein, the terms “product receiving portion” and “membranereceiving portion” are used interchangeably, with it being understoodthat both terms refer to a region of the base of a support assembly ormembrane product package as disclosed herein that is configured toengage a surface of a biological product (e.g., membrane) as disclosedherein and to cooperate with the cover of the support assembly tosupport the biological product in an operative position.

As used herein, the term “support assembly” generally refers to thecombination of a base and a cover as disclosed herein.

As used herein, the term “membrane product package” generally refers tothe combination of a base, a cover, and a membrane positioned betweenthe base and the cover, as further disclosed herein.

As used herein, the term “traction-creating feature” refers to astructural feature of the product receiving portion of a base asdisclosed herein that exhibits a high affinity for a biological product(e.g., membrane) and/or that promotes adhesion, coupling, or otheroperative contact between a biological product (e.g., membrane) and theproduct receiving portion as further disclosed herein, which can producesurface traction between the top surface of the product receivingportion of the base and the product (e.g., membrane) to preventundesired movement of the product relative to the base. Exemplarytraction-creating features include a rough surface; a plurality ofperforations; a surface comprising a plurality of channels; a surfacecomprising a plurality of grooves; a surface comprising a plurality ofindentations; or a surface comprising a plurality of porations.Optionally, in use, it is contemplated that the perforations, channels,grooves, indentations, porations, and other void spaces can effectivelycreate a suction force that adheres the biological product (e.g.,membrane) to the product receiving portion, whereas the rough surfacesdisclosed herein can mechanically (e.g., frictionally) engage the bottomsurface of the biological product (e.g., membrane) to resist movement ofthe product relative to the base. Examples of such a “rough surface”include, for example and without limitation, an abraded surface, ascratched surface, an uneven surface, a gritty-type surface (yet,preferably free or substantially free of loose particulate), or a bumpysurface, among others. Optionally, it is contemplated that the productreceiving portion can comprise a combination of different rough surfacesthat cooperate to define the product receiving portion.

Overview

The present technology provides compositions, articles of manufacture,devices, systems and methods of utilizing each for the protection oftissues, membranes, or graft materials, for example, during manufacture,processing, cryopreservation, storage and transport to the health careprovider/health care provider site. Further, the present technologyprovides compositions, articles of manufacture, devices, systems andmethods of utilizing each for the delivery or application of tissues,membranes, other biological materials and grafts to a human or animal inneed thereof. In particular, the present technology providescompositions, articles of manufacture, devices, systems and methods ofutilizing each for the treatment of wounds, tissue defects or membranedefects or injuries in a human or animal. Further, the presenttechnology provides compositions, articles of manufacture, devices,systems and methods of utilizing each for the preparation, storage,transportation and delivery of tissue, membrane, grafts or otherbiological products for others uses, including but not limited todiagnostics, experimental testing and the like.

The present technology in at least some aspects and embodimentscomprises a device, composition, article of manufacture or system(namely in the form of packaging) comprising at least a base and a coversuitable for use with a biological membrane, tissue or graft (or otherbiological material) that can support, stabilize and protect suchbiological materials during manufacture, storage, transportation anddelivery/application (preferably by a health care provider) to an enduser (i.e., a human or animal patient) or wound. Generally, the presenttechnology also comprises at least one location wherein the base andcover are in communication with one another. Such communication betweenthe base and the cover can optionally be separate from the communicationthat the base and the cover have with a tissue, membrane, graft or otherbiological material as disclosed herein. Further, it should beappreciated by those skilled in the art that the present technology alsoprovides devices, compositions, articles of manufacture and systems thatcan be used multi-functionally as a carrier for a membrane, tissue,other biological material or graft during the resultant packaging's orpackaging system's manufacture and storage, including for example,during cryopreservation and thawing of the associated membrane, tissue,graft, or other biological materials. Moreover, the present technologyprovides at least one device, article of manufacture, composition and/orsystem that allow for a desired orientation (i.e., directionality,spatial arrangement, and/or positioning) of the membrane, tissue,biological material or graft material that is temporarily applied,supported, associated, or affixed thereto in some manner or fashion.

Disclosed herein with reference to FIGS. 1A-8 is a support assembly forsupporting a biological product (e.g., membrane) 20 in an operativeposition. In exemplary aspects, the support assembly can comprise a base30 and a cover 60.

In one aspect, and with reference to FIGS. 1A, 2A, 3A, 4A, and 5A-5C,the base 30 can have a longitudinal axis 32 and comprise a product(e.g., membrane) receiving portion 40. In this aspect, the productreceiving portion 40 can have a top surface 42 and an opposed bottomsurface 44 that are spaced apart relative to a vertical axis 34 that isperpendicular to the longitudinal axis 32 of the base 30. In exemplaryaspects, the product receiving portion 40 of the base 30 can comprise atleast one traction-creating feature that is configured to promotesurface traction between a product (e.g., membrane) and the base.Optionally, in one exemplary aspect, the traction-creating feature ofthe product receiving portion 40 can be selected from the groupconsisting of (i) a rough top surface 80 as further disclosed herein(see FIG. 9); and (ii) a plurality of perforations 46 that extendbetween the top and bottom surfaces 42, 44 of the product receivingportion. Thus, in some optional aspects, the product receiving portion40 can comprise a rough top surface 80, while in other optional aspects,the product receiving portion 40 can define a plurality of perforations46.

In another aspect, and with reference to FIGS. 1B, 2B, 3B, 4B, and 5A,the cover 60 can have a longitudinal axis 62, a top surface 64, and anopposed bottom surface 66. In this aspect, and as further disclosedherein, the cover 60 can be configured for releasable coupling(optionally, releasable attachment) to the base 30 in a product-coveringposition. As shown in FIG. 5C, in the product-covering position, thecover 60 can overlie the product receiving portion 40 of the base 30 andany product (e.g., membrane) 20 positioned over the product receivingportion. In exemplary aspects, the base 30 and the cover 60 areconfigured to cooperate to support the biological product 20 in theoperative position. With reference to FIGS. 5A-5C, in the operativeposition, the biological product 20 is positioned in engagement with atleast a portion of the top surface 42 of the product receiving portion40 of the base 30 and at least a portion of the bottom surface 66 of thecover 60. As further disclosed herein, it is contemplated that theoperative position can correspond to a desired orientation of theproduct 20, such as, for example and without limitation, an advantageousorientation for application of the product 20 to a human or animalpatient.

In a further aspect, the base 30 can further comprise a handling portion50 that is positioned adjacent to the product receiving portion 40relative to the longitudinal axis 32 of the base. Optionally, inexemplary aspects, in the product-covering position, the cover 60 doesnot overlap with the handling portion 50 of the base 30. In furtheroptional aspects, the handling portion 50 can comprise a tab. In anexemplary aspect, the handling portion 50 of the base 30 can have alongitudinal length 51 and a width, wherein the product receivingportion 40 of the base has a longitudinal length 41 and a width, andwherein the width of the product receiving portion is equal to the widthof the handling portion (see FIG. 2A, showing the base 30 having aconstant width 38). Optionally, in some aspects, the longitudinal length41 of the product receiving portion 40 can be greater than thelongitudinal length 51 of the handling portion 50. Optionally, in otheraspects, the longitudinal length 41 of the product receiving portion 40can be less than the longitudinal length 51 of the handling portion 50.In further optional aspects, the longitudinal length 41 of the productreceiving portion 40 can be substantially equal to the longitudinallength 51 of the handling portion 50. As one will appreciate, incombination, the longitudinal length 41 of the product receiving portion40 and the longitudinal length 51 of the handling portion 50 can definea longitudinal length 36 of the base 30. However, in some optionalaspects, and as further disclosed herein, it is contemplated that theproduct receiving portion 40 can extend along substantially the entirelongitudinal length 36 of the base 30, in which case the longitudinallength 41 of the product receiving portion will be substantially equalto the longitudinal length of the base.

Optionally, in an additional aspect, in the product-covering position,the longitudinal axis 62 of the cover 60 can be positioned insubstantial alignment with the longitudinal axis 32 of the base 30. Inanother aspect, the cover 60 can have a longitudinal length 70 and awidth 72. Optionally, in this aspect, the longitudinal length 70 of thecover 60 can be substantially equal to the longitudinal length 41 of theproduct receiving portion 40. Optionally, it is further contemplatedthat the width 72 of the cover can be substantially equal to the width38 of the product receiving portion.

In a further aspect, and with reference to FIGS. 1A-1B, the cover 60 canhave a plurality of corners 68. Optionally, in this aspect, at least oneof the corners 68 of the cover is rounded. In exemplary aspects, it iscontemplated that the cover 60 can have four rounded corners 68.However, it is contemplated that the corners 68 can have other sharp ornon-sharp profiles, such, as for example and without limitation, abeveled profile. In further exemplary aspects, it is contemplated thatthe product receiving portion 40 of the base 30 can have two roundedcorners 45. In these aspects, it is contemplated that, in theproduct-covering position, two rounded corners 68 of the cover 60 canoverlie the two rounded corners 45 of the product receiving portion 40of the base 30. It is further contemplated that the handling portion 50of the base 30 can have two corners (optionally, rounded corners) 52that are positioned in opposition to the corners 45 defined by theproduct receiving portion 40.

In exemplary aspects, the plurality of perforations 46 of the productreceiving portion 40 of the base 30 can be substantially evenlydistributed throughout the product receiving portion.

In further exemplary aspects, the plurality of perforations 46 of theproduct receiving portion 40 of the base 30 can be randomly distributedthroughout the product receiving portion.

In an additional aspect, and with reference to FIG. 8, each perforation46 of the plurality of perforations can have a respective diameter 47.Optionally, in exemplary aspects, the diameter 47 of each perforation 46can range from about 0.1 mm to about 5 mm. Optionally, it iscontemplated that the perforations 46 can have substantially equaldiameters. However, it is further contemplated that at least oneperforation 46 of the plurality of perforations can have a diameter 47that is substantially different than the diameter of at least one otherperforation.

In another aspect, and with reference to FIG. 8, each perforation 46 ofthe plurality of perforations can have a respective center point 48.Optionally, in this aspect, it is contemplated that the center points 48of neighboring perforations 46 can be spaced apart by a distance 49ranging from about 0.35 mm to about 10 mm.

In exemplary aspects, and with reference to FIGS. 5A-7B, the disclosedsupport assembly (base 30 and cover 60) can be provided as part of amembrane product package 100. In these aspects, in addition to thesupport assembly, the membrane product package 100 can comprise amembrane 20 positioned in an operative position between the productreceiving portion 40 of the base 30 and the cover 60 (relative to thevertical axis 34). As further disclosed herein, the membrane 20 can havean upper surface 26 and an opposed lower surface 28. It is contemplatedthat the membrane 20 can be positioned in engagement with at least aportion of the top surface 42 of the product receiving portion 40 of thebase 30 and at least a portion of the bottom surface 66 of the cover 60.

In one aspect, the lower surface 28 of the membrane 20 can be attachedto the top surface 42 of the product receiving portion 40 of the base 30at least one attachment point 22. Optionally, in exemplary aspects, thelower surface 28 of the membrane 20 can be attached to the top surface42 of the product receiving portion 40 of the base 30 at least threeattachment points 22. Optionally, in further exemplary aspects, thelower surface 28 of the membrane 20 can be attached to the top surface42 of the product receiving portion 40 of the base 30 at least fiveattachment points 22. Optionally, it is contemplated that the attachmentpoints 22 can be cauterization points (where the product receivingportion 40 and the membrane 20 are cauterized together).

In one aspect, the cover 60 can be attached to the upper surface 26 ofthe membrane 20 at least one attachment point 24. Optionally, inexemplary aspects, the cover 60 can be attached to the upper surface 26of the membrane 20 at least two attachment points 24. Optionally, infurther exemplary aspects, the cover 60 can be attached to the uppersurface 26 of the membrane 20 at least three attachment points 24.Optionally, it is contemplated that the attachment points 24 can becauterization points (where the cover 60 and the membrane 20 arecauterized together).

Optionally, at least one attachment point 24 where the cover 60 isattached to the membrane 20 can overlie and/or substantially correspondto an attachment point 22 where the product receiving portion 40 of thebase 30 is attached to the membrane 20. At these attachment points, itis contemplated that the base 30, the membrane 20, and the cover 60 canbe secured together.

In addition to, or alternatively to, the attachment of the membrane 20to the product receiving portion 40 and/or the cover 60, the top surfaceof the product receiving portion of the base can be directly attached tothe cover at least one attachment point. Optionally, in exemplaryaspects, the top surface 42 of the product receiving portion 40 of thebase 30 can be attached to the cover 60 at least three attachmentpoints. In these arrangements, it is contemplated that the membrane canhave a length and a width that are less than the longitudinal length andthe width of the product receiving portion 40 and the cover 60 tothereby define a peripheral edge region around the membrane 20, and atleast one attachment point (where the product receiving portion isdirectly attached to the cover) can be positioned in the peripheral edgeregion and spaced from an outer edge of the membrane.

In exemplary aspects, the membrane 20 can be a natural membrane, suchas, for example and without limitation, a placental tissue product.Optionally, in one aspect, the membrane 20 can be a chorionic membraneproduct. Optionally, in a further aspect, the membrane can be anamniotic membrane product.

In further exemplary aspects, the membrane 20 can be a syntheticmembrane.

In still further exemplary aspects, it is contemplated that the membrane20 and the top surface 42 of the product receiving portion 40 of thebase 30 can have sufficient surface traction to maintain the membrane inthe operative position following removal of the cover 60 from the base.

In additional exemplary aspects, the membrane 20 and the top surface 42of the product receiving portion 40 of the base 30 can have a firstsurface traction. In these aspects, it is contemplated that the membrane20 and the cover 60 can have a second surface traction that is lowerthan (less than) the first surface traction.

In exemplary aspects, and with reference to FIGS. 5A-5C, a method ofproducing a membrane product package as disclosed herein is provided. Inthese aspects, the method can comprise positioning a membrane in anoperative position between the product receiving portion of the base andthe cover of the support assembly. In these aspects, the membrane can bepositioned in engagement with at least a portion of the top surface ofthe product receiving portion of the base and at least a portion of thebottom surface of the cover.

Optionally, the step of positioning the membrane in the operativeposition can comprise attaching the membrane to the top surface of theproduct receiving portion at a plurality of attachment points asdisclosed herein. It is further contemplated that the step ofpositioning the membrane in the operative position can further compriseattaching the membrane to the cover at a plurality of attachments pointsas disclosed herein.

Optionally, in further aspects, the method can further comprisepositioning the base, the membrane, and the cover within acryopreservation solution. In these aspects, when the traction-creatingfeature of the product receiving portion of the base comprises aplurality of perforations as disclosed herein, the plurality ofperforations can provide contact between the membrane and thecryopreservation solution sufficient to cryopreserve the membrane.

In additional exemplary aspects, a method of applying a membrane isdisclosed. In these aspects, the method can comprise removing the coverfrom a membrane product package as disclosed herein to expose a topsurface of the membrane. In another aspect, the method can furthercomprise disengaging the membrane from the top surface of the productreceiving portion of the base. In a further aspect, the method canfurther comprise selectively applying the membrane to a desired locationon a human or animal patient.

In further exemplary aspects, a kit for repairing a tissue defect isdisclosed. In these aspects, the kit can comprise a membrane productpackage 100 as disclosed herein. In additional optional aspects, the kitcan further comprise a container (e.g., a bag) that encloses themembrane product package. In these aspects, the container can beselectively opened to provide access to the membrane product package. Infurther optional aspects, the kit can further comprise instructions forapplying the membrane of the membrane product package to repair thetissue defect. In still further optional aspects, the kit can furthercomprise a cryopreservation solution. Optionally, in other aspects, thekit can further comprise a basin configured to receive the membraneproduct package. In these aspects, it is contemplated that the basin canserve as a wash basin and/or thawing basin for the membrane productpackage. In still another aspect, the kit can optionally comprisescissors. In yet another optional aspect, the kit can comprise tweezers.

Further exemplary aspects of the disclosed concepts are provided in thefollowing sections of the specification.

The Base

a. Product Membrane Receiving Portion

As shown in FIGS. 1A, 2A, 3A, 5A-5C, 6A, 7A, and 9, the base 30 of thepresently described technology comprises at least one receiving portion40. The receiving portion 40 is capable of receiving a biologicalproduct, material, or composition 20. Such materials or compositions mayinclude, for example, membranes, tissues, graft materials, and the like.Throughout the remainder of the specification and appended claims, thebiological product will generally be referred to as a “membrane,” andthe receiving portion 40 shall be referred to interchangeably as eitherthe “product receiving portion” or the “membrane receiving portion.” Itshould be appreciated by those skilled in the art, however, that theterm encompasses and contemplates the receipt and engagement of otherbiological materials such as tissues, other biological materials andgrafts. The membrane receiving portion 40 is a portion of the base 30(of the present technology) that contacts the membrane 20. The membranereceiving portion 40 of the present technology also can comprise atleast one traction-creating feature. As further disclosed herein, thetraction-creating feature provides a surface which, when in contact withthe membrane 20, provides sufficient surface traction such that themembrane remains sufficiently but temporarily attached to the base 30and prevents, for example, curling or detachment of the membrane duringmanufacture, storage, transport and handling prior to final removal fromthe packaging or packaging system and application to an end user or foran end use application (e.g., wound treatment, diagnostic testing orexperimental/analytical laboratory usages). Thus, the traction-creatingfeature (e.g., a structured surface) provides sufficient support,attachment/connection and/or stabilization of the membrane 20 inconjunction with the base 30 when applied thereto. This is unexpectedsince the packaging device, composition, article of manufacture orsystem of the present technology itself (e.g., the support assemblydisclosed herein), not the membrane, tissue or graft material, providessuch outcomes, especially during each of the phases of preparing,storing, transporting, handling, and administering of the end product.It is also unexpected that the presently described technology canprovide such outcomes while still allowing the end user to size, shapeand finally apply the end product to the patient (human or animal) in aconvenient manner without significant waste, destruction, damage,injury, or other negative outcome to the membrane to be applied.

As further described herein, the traction-creating features may be anysuitable surface feature that provides the necessary surface tractionwhen in contact with the membrane, tissue, biological material or graftmaterial. The surface traction necessary to maintain contact with themembrane, tissue, or graft (or other biological material to bedelivered) will depend upon the composition of the membrane, tissue,graft or other biological material to be applied, temporarily affixed orattached in some non-permanent manner to the membrane receiving portion40. The type of material used to form or make the base 30 will alsoaffect the surface traction necessary to maintain contact between themembrane, tissue, graft or other biological material with thetraction-creating feature of the membrane receiving portion of the base.Thus, it should be appreciated by those skilled in the art that thesurface traction depends on a number of factors, including the type ofbase material selected, the traction-creating features of the membranereceiving portion (including, for example, the perforation, channel,groove, indentation pattern, or other pattern or surface typeselected/desired), and the type of membrane. In at least one embodimentof the present technology, a sufficient surface traction ischaracterized by a package (i.e., a device, a composition, an article ofmanufacture) or packaging system of the present technology having thefollowing features: 1) at least one membrane that does not spontaneouslydetach from the base (or a selected portion, segment or part of thebase) when submerged in a medium (e.g., a biological medium, including abiological solution) and 2) the ability of the membrane to slide fromthe base without ripping, tearing or damage to the membrane when removedfrom the packaging or packaging system and then subsequently applied tothe wound or tissue defect of the human or animal to be treated.

Other suitable methods of testing surface traction sufficient for thepurposes of practicing the present technology may be determined byequipment and methodology known conventionally. For example, asufficient surface traction can be determined instrumentally via anInstron measurement device commercially available from Instron,Incorporated of Norwood, Mass. (a manufacturer of surface tractiontesting equipment designed to evaluate the mechanical properties ofmaterials and components (www.instron.us/)). Surface traction in someinstances is also known as sliding frictional force. Sliding frictionalforce is understood and can be determined by one skilled in the art, forexample, see Sliding Friction: Physical Principles and Applications(NanoScience and Technology) by Bo Persson (Jun. 21, 2000) 2^(nd)edition, Springer (ISBN-10: 3540671927|ISBN-13: 978-3540671923), andAdvances in Soft Matter Mechanics by Shaofan Li, and Bohua Sun (2012),(ISBN: 978-3-642-19372-9 (Print) 978-3-642-19373-6 (Online)),incorporated by reference in their entireties.

Traction-creating features (e.g., structured surfaces) can include, butare not limited to, for example, a rough surface (e.g., an unevensurface, a scratched surface, and the like), a surface comprising aplurality of perforations or porations, a surface comprising a pluralityof channels (a channeled surface), a surface comprising a plurality ofgrooves (a grooved surface), or a surface comprising a plurality ofindentations (an indented surface), among others. Combinations of suchsurfaces can also be utilized. In some instances, the traction-creatingfeatures comprise at least one perforation, at least one channel, atleast one groove, at least one indentation, and in some instances, theat least one perforation, at least one channel, at least one groove orat least one indentation is a complex pattern or design. In oneexemplary aspect, the traction-creating feature can comprise asandpaper-roughened surface.

Further, a variety of patterns, designs or shapes of varioustraction-creating features (e.g., structured surfaces) can also beutilized in the practice of the presently described technology. Forexample, the traction-creating feature (e.g., structured surface) may bea circular pattern of perforations, alternatively, a square pattern ofperforations and the like. Further designs, shapes, and patternssuitable for use in the practice of the present technology areillustrated in FIGS. 1A-9. It should be appreciated by those skilled inthe art that any pattern, design or shape may be utilized as long as asufficient surface traction between the traction-creating feature andthe membrane, tissue, graft or other biological material can be achievedsuch that the membrane, tissue, graft, or other biological material isstable and supported during manufacture, storage, transport and handlingprior to final application of the membrane to the end user (or for itsuse in an end application such as diagnostic or analytical testing).Yet, the sufficient surface traction (and associated attraction,affinity, and/or adhesion) is only temporary such that the end user canremove the membrane, tissue, graft, or other biological material forfinal application to the human or animal patient (or for final endapplication usage) without significant negative outcomes such ascurling, self-adherence, damage, injury and the like. In someembodiments of the present technology, the traction-creating feature(e.g., structured surface) can be irregular, continuous, discontinuous,symmetrical, dissymmetrical in design, shape or pattern, or comprise acombination of different types of traction-creating features (e.g.,structured surfaces).

During storage, such as cryopreservation storage, it should beappreciated by those skilled in the art that the traction-creatingfeature (e.g., structured surface) of the membrane receiving portionprovides enough or sufficient surface traction such that the membrane isable to temporarily adhere or remain attached or connected to the baseand is not significantly dislodged from the base when a cryopreservationsolution is introduced into the packaging device, composition, articleof manufacture or system of the present technology. It has beensurprisingly found that the present technology allows the membrane,tissue, graft or other biological material to remain temporarilyadhered, attached or connected to the membrane receiving proportionsufficient to withstand shear fluid force that is typically producedwhen a cryopreservation solution or other solution or liquid material isintroduced into the packaging device, article of manufacture,composition, or system. The fluid may be introduced, for example, into abag or other suitable container that can be part of the device,composition, article of manufacture, or system of the present technologythat may hold the base/membrane/cover configuration therein orthereupon. Again, it should be appreciated by those skilled in the artthat the present technology via the traction-creating feature (e.g.,structured surface) of the membrane receiving portion of the base (aloneor alternatively in combination or further communication with thecover), provides sufficient surface traction with the membrane, tissue,graft or other biological material to stabilize, support, and totemporarily hold in place that membrane while withstanding freezingprocedures, shipping, storage, handling and thawing procedures prior tofinal application to a wound or tissue defect.

The membrane receiving portion of the present technology may span theentire length of the base or may span only a portion, section, part orsegment of the base. In some aspects, for example, the membranereceiving portion can span at least 30% of the length of the base. Inother aspects, the membrane receiving portion can span at least 40% ofthe length of the base. In additional aspects, the membrane receivingportion can span at least 50% of the length of the base 30. In stillfurther exemplary aspects, the membrane receiving portion can span atleast 60% of the length of the base 30. In still further exemplaryaspects, the membrane receiving portion can span at least 70% of thelength of the base 30. In still further exemplary aspects, the membranereceiving portion can span at least 80% of the length of the base 30. Instill further exemplary aspects, the membrane receiving portion can spanat least 90% of the length of the base 30. In still further exemplaryaspects, the membrane receiving portion can span at least 95% of thelength of the base 30. In some embodiments, the membrane receivingportion spans about 95% of the length of the base. In other embodiments,for example, the membrane receiving portion spans about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%,about 99%, or about 100% of the length of the base, and it should beappreciated that such spans can include increments and percentages inbetween (for example, 70%, 71%, 71.5%, 72%, 72.5%, 73%, among others).

The term “plurality” when used to describe a plurality of perforations,a plurality of channels, a plurality of grooves, or a plurality ofindentations, for example, refers to a sufficient number of suchperforations, channels, grooves, indentations and the like beingdistributed throughout the membrane receiving portion so as to provide asufficient tension or surface traction for the graft, tissue, membraneor other biological material to temporarily adhere, connect or attach tothe base and withstand the processing and handling during manufacture,transport, storage, handling and final application to a wound or tissuedefect.

b. Perforation of the Membrane Receiving Portion

In some embodiments of the present technology, and with reference toFIGS. 1A, 2A, 3A, and 5A-5C, the membrane receiving portion 40 comprisesa plurality of perforations 46 and/or porations. In some embodiments,the membrane receiving portion 40 may comprise at least one perforation46. The at least one perforation may be a complex pattern or design.Optionally, each perforation 46 can be a small hole within a material(e.g., the membrane receiving portion of the base). It is contemplatedthat the perforations 46 can be formed by any suitable means in the art.Continuous perforated or microperforated sheets for use in the practiceof the present technology may be prepared by any conventional methodknown in the art utilizing a substrate sufficient and consistent withthe practice and intentions of the presently described technology totemporarily adhere, attach, or connect the membrane 20 while providingsupport as well. Suitable means for perforating the base 30 (or membranereceiving portion 40 of the base) can include, but are not limited to,mechanical perforation devices such as suitably arranged punchingmachines, thermal or ultraviolet lasers operating in a desired frequencyband, rotary pinned perforation rollers, a die and punch set, a vacuum,a needle or water jet perforation device or system, hot pins, anembossing device or system and any combinations thereof, among others.

The plurality of perforations (or porations) 46 may also comprise ashape, design, or pattern or may be randomly orientated within themembrane receiving portion 40 of the base 30. In still furtherembodiments, the plurality of perforations 46 are evenly distributedacross the membrane receiving portion 40. The perforations 46 may besimply ordered or may be arranged according to complex sequences. Thepattern of the plurality of perforations 46 may comprise, for example, agrid pattern (e.g., a series of rows and columns). In some embodimentsof the present technology, the size of each perforation 46 can be fromabout 0.1 mm to about 5 mm. Suitably, the perforations 46 can have adiameter (maximum width) of about 0.2 mm, about 0.3 mm, about 0.4 mm,about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm,about 1 mm, about 1.1 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm,about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm,about 2.0 mm, about 2.1 mm, about 2.2 mm, about 2.3 mm, about 2.4 mm,about 2.5 mm, about 2.6 mm, about 2.7 mm, about 2.8 mm, about 2.9 mm,about 3.0 mm, 3.1 mm, about 3.2 mm, about 3.3 mm, about 3.4 mm, about3.5 mm, about 3.6 mm, about 3.7 mm, about 3.8 mm, about 3.9 mm, about4.0 mm, about 4.1 mm, about 4.2 mm, about 4.3 mm, about 4.4 mm, about4.5 mm, about 4.6 mm, about 4.7 mm, 4.8 mm, about 4.9 mm and about 5.0mm and any increments between, including increments from between about0.01 mm to about 0.1 mm. In further aspects and embodiments of thepresent technology, the perforation size can preferably range from about0.1 mm to about 20.3 mm, and spacing can be about 0.35 mm to about 20 mmcenter to center. Optionally, in some exemplary aspects, the diameter ofat least one perforation 46 can be different (less than or greater than)the diameter of at least one other perforation of the plurality ofperforations.

The number and size of the perforations 46 depends upon the material orsubstrate of which the membrane receiving portion 46 is made from, thetype of membrane 20, tissue, graft or other biological material adhered,attached, connected or associated (all temporarily) with the membranereceiving portion, and the surface traction that is sufficient tomaintain the membrane, tissue, graft and/or other biological materialtemporarily on, onto, connected to, attached to, adhered to and the liketo the base 30 during processing, storing, transporting, and handling.Additionally, the perforations (or porations) 46 may be any geometricalor non-geometrical shape. Suitable shapes include, but are not limitedto, circular, oval, rectangular, square, diamond, trapezoidal, star,hexagonal, octagonal, semi-circular, crescent, ellipse or a combinationthereof. Perforations (or porations) 46 may also be a section or part ofa shape, such as a half star or half crescent. It should be understoodby those skilled in the art that one or more shapes may be used in anycombination as well.

The center-to-center distance 49 between adjacent (neighboring)perforations (or porations) 46 on the base 30 (or membrane receivingportion 40 of the base) depends upon the size and number of perforations(or porations) 46 that can be distributed throughout the area selected(for example, the size of the membrane receiving portion) and theoverall selected size of the base, itself. The size and number ofperforations (or porations) 46 permissible in the base 30 (or membranereceiving portion 40 of the base), in turn, depend upon the effect ofthe same upon the physical properties of the base, the cover 60, and thestabilizing, temporarily connective and protective functions served bythe base and structure of the perforations with respect to theassociated membrane 20, tissue, graft and/or other biologicalmaterial(s). For example, the distance 49 between perforations 46 asmeasured from the center of one perforation to the center of anotherperforation may be from about 0.3 to about 10 mm, alternatively fromabout 0.35 mm to about 10 mm, alternatively from about 0.35 mm to about5 mm, alternatively from about 1 mm to about 5 mm, alternatively fromabout 4 mm to about 10 mm, and any increments and distances in between,including increments from about 0.01 mm to about 0.1 mm. Preferably, thedistance 49 between perforations (or porations) 46 is from about 1 mm toabout 10 mm, more preferably about 3 mm to about 5 mm, such as 3 mm or 4mm.

While not wanting to be bound by any particular theory, it has beenobserved and discovered in the practice of the present technology thatthe smaller the membrane 20 (tissue, graft, or other biologicalmaterial(s)) to be attached to the base 30 (or membrane receivingportion 40 of the base), the smaller the perforations (or porations) 46can be within the base (or membrane receiving portion of the base).Conversely, the larger the membrane 20 to be attached to the base 30 (ormembrane receiving portion 40 of the base), larger perforations (orporations) 46 can be utilized and the farther apart the perforations (orporations) can be spaced as well. For example, for a 7.5 cm×15 cm (7.5cm length×15 cm width) membrane, the perforations may be about 5 mm indiameter and about 10 mm apart (as measured center to center). For amembrane that is approximately 1.5 cm×2 cm, the perforations may beabout 1 mm diameter and about 4 mm apart center to center. In someembodiments of the present technology, for a membrane that isapproximately 5 cm×5 cm or 3 cm×4 cm, the perforations may be about 1 mmdiameter and about 4 mm apart center to center. In other embodiments,for an approximately 2 cm×3 cm membrane, the perforations may be about 1mm diameter and about 3 mm apart center to center. It should beappreciated by those skilled in the art that such examples are forillustrative purposes only and are not to be considered exhaustive. Insome instances, not to be bound by any particular theory, for membranesizes over 100 cm² the size of perforations may be increased up to fivefold and the distance between the perforations may be increased by 2-3fold.

Preferably, the method of forming perforations (or porations) should notcreate microparticles or other impurities or contaminants (orpollutants) that are permanently stained on the base and cannot beremoved, or would otherwise be detrimental to the membrane. Further, theperforated (or porated) base (or membrane receiving portion thereof)should be free of oil or other chemicals, materials, impurities,pollutants, contaminants or substances that may interfere with theperformance of the base and the membrane receiving portion according tothe practice of the present technology, or would otherwise not bebiocompatible or would be detrimental to the membrane. Moreover, thebase and membrane receiving portion (however modified, for example, viaperforating, grooving, channeling, etc.) should be able to maintain asufficient cleanliness to protect and/or maintain the cellular viabilityof the associated membrane, tissue, graft and/or other biologicalmaterials temporarily associated therewith, maintain integrity of theassociated membrane, tissue, graft and/or other biological materialstemporarily associated therewith, and/or provide safety for the human oranimal being treated (e.g., comply with safety regulations). In doingso, a detrimental response by a patient to the membrane, tissue, graftand/or other biological material(s) can be reduced or prevented whenprovided to patients, (e.g., when applied to or transplanted on orwithin various areas of a patient(s) such as a wound or tissue defect).

c. Grooves/Channels/Indentations, Etc.

In some embodiments of the present technology, the membrane receivingportion 40 may comprise a plurality of grooves. In some embodiments, themembrane receiving portion 40 may comprise at least one groove, whereinin some instances, the at least one groove is a complex pattern orshape. The grooves can span the entire length of the membrane receivingportion or any segment, portion, or part thereof. The grooves may bediscontinuous or continuous over the entire length or portion (orsegment or part) of the membrane receiving portion of the base. Thegrooves can be orientated in parallel or in a perpendicularconformation, or a combination thereof. The grooves may be evenlydistributed or randomly distributed over the respective length, segment,portion or part of the membrane receiving portion. The grooves shouldcover a sufficient area of the membrane receiving portion to provide asufficient surface traction such that the membrane, tissue, graft and/orother biological material associated therewith temporarily adheres,connects, or attaches to the membrane receiving portion (or base) forpurposes of stabilization, temporary connection and protection duringmanufacture, handling, storage, transport and final application orusage.

In other embodiments of the present technology, the membrane receivingportion 40 may comprise a plurality of channels. The channels may bediscontinuous or continuous over the entire length of the membranereceiving portion, or alternatively a portion, part, or segment thereof.The channels may be evenly distributed or randomly over the membranereceiving portion. The channels can be orientated in a parallelconformation to one another, a perpendicular conformation to oneanother, or a combination thereof. The channels preferably cover asufficient area of the membrane receiving portion of the base to providesufficient surface traction such that the membrane temporarily adheres,connects or attaches to the membrane receiving portion duringmanufacturing, handling, storage and final application or usage.

In still other embodiments, the membrane receiving portion 40 of thepresent technology can comprise a plurality of indentations. Theindentations may be evenly distributed over the entire length, portionor segment of the membrane receiving portion of the base. The channelsmay be randomly or evenly distributed over the entire length, portion orsegment of the membrane receiving portion. The indentations may also bearranged in rows and other patterns, designs or configurations. The rowsmay be parallel or intersecting over the entire (or alternatively aportion, part, or segment of the) length, of the membrane receivingportion of the base. The indentations should cover a sufficient area ofthe membrane receiving portion of the base to provide sufficient surfacetraction such that a membrane, tissue, graft, or other biologicalmaterial(s) sufficiently but temporarily adheres, connects or attachesto the base during manufacturing, handling, storage, transit and finalapplication or usage.

d. The Membrane Receiving Portion Surface

In some embodiments of the present technology, and with reference toFIG. 9, the membrane receiving portion 40 of the base is a rough surface80, preferably a rough plastic surface. However, it should beappreciated that other materials suitable for use to practice thepresently described technology are also envisaged. Suitable means ofmaking a rough surface include, but are not limited to, sanding,chemical alteration, 3-D printing, abrasive blasting, and other methodsknown to one skilled in the art. The rough surface 80 should provide asufficient surface traction such that the membrane, tissue, graft, orother biological material(s) temporarily adheres, connects, or attachesto the membrane receiving portion of the base during manufacturing,handling, storage, transit and final application or usage, but not toostrongly so as to not be able to be easily removed and applied (e.g., toa wound or tissue defect or for use in a further procedure).

In other embodiments, the membrane receiving portion 40 may exhibit ascratched surface. Again, the scratched surface should provide asufficient surface traction such that the membrane, tissue, graft, orother biological material(s) temporarily adheres, connects, or attachesto the membrane receiving portion of the base during handling, storage,transit and application, but not too strongly so as to not be able to beeasily removed and applied (e.g., to a wound or tissue defect or for usein a further procedure).

e. Handling Portion

In some aspects and embodiments of the present technology, and withreference to FIGS. 1A, 2A, 3A, 5A-5C, 6A, 7A, and 9, the base 30 furthercomprises at least one handling portion 50. The handling portion 50 maybe, for example, adjacent to the membrane receiving portion 40. Thehandling portion 50 provides a region of the device, system, article ofmanufacture, or composition of the present technology that can behandled or operated by an individual without causing damage, injury toor significant waste, disorientation, or negative outcomes to themembrane 20 (tissue, graft, or biological material(s)) applied thereto.This reduces direct contact with the membrane 20 and a handler, whichreduces damage to the membrane (among other things) and maintains, inthe case of living membranes, cellular viability, and other attendantbeneficial biological properties and functions when applied to a patientor utilized in other therapeutic, diagnostic, analytical and/orexperimental laboratory manners.

In still further embodiments, and with reference to FIGS. 6A and 7A, thehandling portion 50 can also contain a marker, label or designation 55for orientation and/or application. This allows for the indication ofthe directionality, spatial arrangement or proper application of amembrane 20 (e.g., tissue, graft, or other biological materials)temporarily applied to the membrane receiving portion 40 of the base 50,which can be maintained throughout preparation, cryopreservation,storage, transport, thawing and final application to a patient or otherusage. The marker 55 for orientation may be any symbol that can indicatea proper orientation, proper application or usage, and/ordirectionality. For example, the marker 55 can be a name, a trade namefor the packaging product (kit or system), an arrow, at least one wordor a letter, a directional symbol, or other suitable alternatives. Toillustrate, suitable letters that require orientation, for example,include but are not limited to B, C, D, E, F, G, J, K, N, P, Q, R, S,and/or Z. To further illustrate, suitable orientation markers 55 may bethe name of the product, for example Grafix PRIME® or Grafix CORE® ownedby Osiris Therapeutics, Inc. of Columbia, Md. In other embodiments, themarker 55 may be a word or phrase, for example “top,” “this side up,”face,” “forward,” “product,” “handle side”, “keep this side up.” One ofskill in the art shall appreciate additional alternatives that canprovide the orientation, proper application, and/or directionality ofthe device, composition, article of manufacture, or system of thepresent technology. In some embodiments, the label or marking 55 may bea colored letter, square, block or edge. In doing so, the device,composition, article of manufacture, or system of the present technologyprovides the advantage of informing the handler of the properorientation placement of at least one membrane 20 (e.g., tissue, graft,or other biological material) on, onto, or upon the membrane receivingportion 40 of the base 30. Further, the marker 55 for orientation alsoprovides information to a handler (e.g., a health care provider)regarding the proper orientation of the device, composition, article ofmanufacture, or system of the present technology (e.g., the supportassembly or membrane product package 100 disclosed herein) prior to themembrane, tissue, graft, or other biological material(s) being appliedto the patient. Thus, it should be appreciated by those skilled in theart that any shape, symbol, color, design, pattern and the like may beutilized and are envisaged in the practice of this aspect andembodiments of the present technology.

Optionally, in additional embodiments, the cover 60 comprises at leastone handling portion. The handling portion of the cover 60 may alsocontain a marker for orientation and/or labeling of the product asdescribed herein. The handling portion of the cover 60 may also be usedfor removal of the cover) during the end application process (e.g., to apatient or usage during a diagnostic procedure, laboratory analysis orsome other usage). In some of these embodiments, the handling portioncan be a grip. The grip may be used to remove the cover that istemporarily adhered, connected or attached from the membrane, tissue,graft, or other biological material(s) and base.

In other embodiments, both the cover 60 and the base 30 both separatelycomprise at least one handling portion or can jointly form at least onehandling portion. In still further embodiments, the base 30 may comprisea first handling portion while the cover 60 may comprise a secondhandling portion that may operate separately or may work cooperativelywith the first handling portion. In other embodiments, the firsthandling portion and the second handling portion are in communicationwith each other. The communication is preferably temporary butsufficient to maintain communication during manufacture, handling,transport and prior to final application or usage. For example, in someembodiments, the second handling portion can overlap at least a part,portion, or segment of the first handling portion.

Additionally, it should also be appreciated that the respective handlingportion (of the base 30 or the cover 60) can be the entire width of thecover and/or the base. Alternatively, the handling portion may compriseonly a portion, segment, or part of the width of the cover and/or base.A portion, segment, or part of the width may be about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95% of the width, and any percentage or width in between. Thehandling portion should be sufficiently sized so as to provide a regionof the cover and/or base to allow a handler to safely handle the device,composition, article of manufacture or system of the present technologywithout causing injury or damage to the membrane, tissue, graft, orother biological material(s) associated therewith. Further, the handlingportion should be sufficiently sized so as to provide ease of handlingby the handler. Moreover, the handling portion should be sufficientlyshaped so as to provide easy of use while providing a more comfortablehandling, such as through the prevention of cuts upon the handlersfingers during handling that can result if the edges of the handlingportion are not smoothed (e.g., rounded or sanded, etc.).

In some embodiments of the present technology, the handling portion isfrom about 1 cm to about 5 cm wide and the same length as the membranereceiving portion 40 of the base 30. Sizing of the handling portiondepends on the size of the membrane receiving portion of the base, andthe handle portion in some optional embodiments does not exceed the sizeof the membrane receiving portion of the base. In other embodiments, thehandling portion is about 1 cm wide, preferably about 2 cm wide,alternatively about 2.5 cm wide, alternatively about 3 cm wide, andspans the entire length or a portion, part, or segment of the entirelength of the membrane receiving portion. In still further embodiments,the handling portion is preferably at least about 1 cm wide.

The Cover

With reference to FIGS. 1B, 2B, 3B, 4B, 5A, 5C, 6B, and 7B, the cover 60of the compositions, devices, articles of manufacture or systems of thepresent technology (e.g., the support assembly and the membrane productpackage 100) can optionally be the same size as the base 30.Alternatively, in other embodiments, the cover 60 is substantially thesame size as the membrane receiving portion 40 of the base 30.Preferably, the cover 60 is of a sufficient size to protect a membrane20 (e.g., tissue, graft or other biological material(s) that istemporarily attached, connected, or adhered to the cover and/or themembrane receiving portion of the base) from shear forces and stressesproduced or faced during processing, storage, transport, handling andfinal application or usage. In the various embodiments of the presenttechnology, the cover 60 can optionally be a single piece of suitablebiocompatible substrate. In some embodiments, the substrate is abiocompatible plastic, biopolymer, biocopolymer or other biocompatiblematerial that does not significantly injure, damage, contaminate orotherwise harm the associated membrane of the present technology.

Shape/Size/Make-Up of the Cover and Base

In some embodiments of the present technology, the base 30 can exhibit ashape that comprises non-sharp corners 45, 52. Optionally, the cornersof the base 30 can be rounded. In still further embodiments, the cover60 can comprise non-sharp corners 68. In some preferred embodiments, thecorners 45, 52, 68 of the cover and/or base are rounded. Non-sharpcorners are preferred for safety and handling of the device, system,article of manufacture, or composition of the present technology duringmanufacturing, storage, transport, or application/usage, and also toprevent tearing or puncturing of the temporarily associated membrane,tissue, graft, or other biological material(s) during removal from thebase and/or cover. It should be appreciated that the base and cover maybe of any desired geometrical shape. In some preferred embodiments, thebase and cover are squares or rectangles, preferably squares orrectangles with rounded corners. However, it is further contemplatedthat the cover and/or base can have a round shape (e.g., a circular orelliptical shape).

It should also be appreciated by those skilled in the art that thedevice, article of manufacture, composition, or system (e.g., thesupport assembly or the membrane product package 100) can exhibit avarying size. In some embodiments, the size of the device, article ofmanufacture, composition or system can be a customizable size for aparticular application which is chosen by the manufacturer or thehandler prior to end application or usage. For example, in someembodiments, the size (longitudinal length×width) can be about 1 cm×1cm, about 1.5 cm×2 cm, about 1.5 cm×1.5 cm, about 2 cm×2 cm, about 2.5cm×2.5 cm, about 2 cm×3 cm, about 2 cm×4 cm, about 2 cm×5 cm, about 3cm×4 cm, about 3 cm×5 cm, about 5 cm×5 cm, about 4 cm×5 cm, about 5 cm×7cm, about 1.5 cm×3.5 cm, about 7.5 cm×15 cm, about 9.5 cm×15 cm, about7.5 cm×17 cm, and other sizes and ranges there between.

In a variety of embodiments of the present technology, the base and/orcover are each individually made of a singular piece of plastic,preferably a biocompatible plastic. Alternatively, in other embodiments,the base and the cover can be made collectively from a singular piece ofplastic, again preferably a biocompatible plastic. For example, in suchembodiments, the singular piece of plastic can be folded such that thebase and the cover are formed and interact with one another, preferablytemporarily. Further, the first side of the base can be continuous witha first side of the cover. Thus, a membrane, tissue, graft, or otherbiological material may be placed between the first side of the base andthe first side of the cover, wherein a second side of the cover isfacing externally to the membrane. In still further embodiments, thebase and the cover may each be or collectively be a single piece ofbiocompatible plastic produced by 3-D printing. In other embodiments,the single piece of plastic for each of the base and cover may havelatches, handles, or other forms of attachment and connection that canconnect the base to the cover and vice versa. For the example, the firstside of the base can be fixedly attached or connected to the first sideof the cover via two interlocking or connecting handles or a hook andlatch enclosure system, for example.

In further embodiments, the base and the cover can be made of, forexample, at least two substrates. In some embodiments, the substratesare two of the same or different biocompatible plastics. In otherembodiments, the base and the cover are comprised of the same type ofsubstrate while in other embodiments the cover and base are made fromdifferent substrates. Thus, one of ordinary skill in the art willappreciate that a variety of substrates can be utilized to make thevarious parts of the presently described technology as long as thesufficient characteristics of the components allow for the support,stabilization, temporary connection or attachment of the selectedmembrane thereto with a sufficient surface traction to prevent injury,damage, detachment of the membrane during manufacturing, storage,transporting, handling and end application or usage. To illustrate, instill further embodiments, the base and the cover are made fromdifferent types of substrates, with the different types of substratesbeing different types of biocompatible plastics, biopolymers,biocopolymers, or other biocompatible materials. More particularlydifferent types of biocompatible plastics, for example, may be a singleplastic composition, a multiple-composition plastic, or layers of abiocompatible plastic(s) selected. As should be appreciated by thoseskilled in the art, further alternatives are also envisaged.

Communication Between the Base and the Cover

In some embodiments of the present technology, and with reference toFIGS. 5A-7B, the device, article of manufacture, system and/orcomposition comprises a base 30, a cover 60, and at least one location(attachment point 22, 24) in which the base and the cover are inoperative communication, which can be direct communication (e.g.,temporary direct communication) or indirect communication (e.g.,temporary indirect communication) through a membrane 20 as disclosedherein. In other embodiments, the base 30 and cover 60 are insufficiently close proximity and are attached to one another and/or to amembrane 20 by temporary or removable attachments or connections.Suitable methods of communication can include, but are not limited to,heat sealing, cauterization, welding, ultrasonic welding, abiocompatible adhesive (preferably an adhesive suitable for use withbiological and/or cellular materials), use of a laser, use of aninterconnecting means, crimping (by heat or mechanically by pressure),crunching, stapling, or clamping, among others. Preferably, theattachment or connections include, but are not limited to, at least onediscrete cauterization point, at least one latch, or at least oneultrasonic welding point by which the cover, membrane (tissue, graft, orother biological material(s)) and base are temporarily connected orattached. To illustrate, a sufficient number of cauterization points(attachment points 24) can be utilized to attach the membrane 20(tissue, graft, or other biological material(s)) to the cover 60 and asufficient number of cauterization points (attachment points 22) can beutilized to attach the membrane 20 (tissue, graft, or other biologicalmaterial(s)) to the base 30 as well. Optionally, when the membrane 20has a smaller length and/or width than the cover 60 and the membranereceiving portion 40 of the base 30, a sufficient number ofcauterization points (or other attachment points) can be utilized toattach the cover directly to the membrane receiving portion, with themembrane being inwardly spaced from such attachment points. Thus, it iscontemplated that the membrane 20 can be secured between the cover 60and the base 30 without directly attaching the cover and/or the base tothe membrane at an attachment point. It should be appreciated by thoseskilled in the art that any number of cauterization points or othertemporary attachments between the membrane (tissue, graft, or otherbiological material(s)) and the cover and/or base can be used in thepractice of the present technology. Further, the level of temporaryattachment can be varied depending upon the type of material used tomake the cover and/or base as well as the type, size, depth (among othervariables) of the membrane, tissue, graft or other biologicalmaterial(s) temporarily attached, connected, or applied thereto (i.e.,temporarily attached or connected to the cover, the base or both). Inexemplary aspects, the attachment points (e.g., cauterization points)can be generally positioned in the outer edge portions of the cover 60,base 30, and membrane 20, thereby minimizing functional damage to thesecomponents.

Focusing upon the connective relationship between the cover and base, insome additional embodiments, the at least one connection or attachmentlocation in which the base and the cover are in communication with oneanother (inclusive or not inclusive of the membrane, tissue, graft, orother biological material(s)) comprises one or more interconnectingmeans or connective devices, connectors and the like. For example, thecommunication can be a female connector and a male connector, whereinthe male connector secures into the female connector to provide asuitable attachment between the base and the cover. The suitableconnection may be disrupted by sufficient force to remove the maleconnector from the female connector. In other embodiments, the femaleconnector can comprise an indentation while the male connector cancomprise a protrusion in a shape suitable to fit or connect within theindentation. Again, these examples are for illustrative purposes onlyand are not an exhaustive exemplary listing.

It should also be appreciated that the number of temporary connection orattachment locations or points between the base and the cover, as wellas the base and/or cover and the membrane (tissue, graft, or otherbiological material(s)) depends on the manufacturing processes andhandling processes utilized as well as the type of membrane (tissue,graft, or other biological material(s)) selected. However, in accordancewith the practice of the present technology, the number of temporaryconnection or attachment locations must provide sufficient traction,adhesion, attachment or connection to withstand multiple stressesexperienced during processing, storage, transport, handling andusage/application. For example, the number of connection or attachmentlocations must be sufficient to ensure the associated membrane, tissue,graft, or other biological material(s) remains temporarily attached,adhered, or connected to the cover and/or base while enduring fluidshear force created during manufacturing of the present technology orits packaging, during cryopreservation and thawing and also duringtransit should the cryomedium potentially thaw prematurely, shift, moveor otherwise develop shear forces and the like that could potentiallyaffect the membrane interacting therewith.

Additionally, as further described herein, the number of connection orattachment locations of the present technology (i.e., those of the coverand the base as well as those of the cover and/or base to the membrane,tissue, graft, or other biological material(s)) must be able towithstand storage (including, for example, cryopreservation (as well asthe step of the inclusion or addition of a cryopreservation mediumprovided during the manufacturing or storage process)). Moreover, theconnection or attachment locations also are preferably capable ofwithstanding temperature changes, including temperature changes rangingfrom 60° C. to −196° C. (+5° C.), alternatively from about 80° C. toabout −196° C.±5° C. (alternatively from about 40° C. to about −196° C.(+5° C.) for a membrane or biological material containing viable cells),among others. Conversely, the connection or attachment locations and/orpoints of the present technology are also preferably capable ofwithstanding the temperature variances and other forces associated withthawing of the device, composition, article of manufacture, or system ofthe present technology. For example, such connection or attachmentlocations preferably are maintained when the device, composition,article of manufacture, or system is unthawed from about −80° C. or−196° C. (+5° C.) to about room temperature. (20° C. to about 25° C.) orabout 40° C. (+5° C.). Further, the connection or attachment locationsand/or points of the present technology described herein also preferablyare capable of withstanding additional handling prior to finalapplication or usage such as during washing of the device, composition,article of manufacture, or system during the performance of a thawingprocedure.

The number of connection or attachment locations will depend upon theconnection or attachment properties of the membrane, tissue, graft orother biological material(s) utilized and the type of material (e.g., aplastic substrate selected) used to form the cover and/or the base. Thenumber of connection or attachment locations should allow for easyremoval of the cover without disturbing the further attachment orconnection between the membrane, tissue, graft, or other biologicalmaterial(s) and the base. Further, the connection(s) or attachment(s)between the base and the membrane (tissue, graft, or other biologicalmaterial(s)) should also be easily broken without tearing or damagingthe membrane (tissue, graft, or other biological material(s)).Additionally, the connection(s) or attachment(s) between the base and/orthe top and the membrane should be such that they do not contaminate,break-off, or pollute the membrane. Moreover, the connection(s) orattachment(s) between the base and the membrane (tissue, graft, or otherbiological material(s)) should allow a handler (e.g., a health careprovider) to easily remove and apply the membrane, tissue, graft orother biological material(s) to an end user (e.g., a patient having awound or tissue defect) or for use in an end use application.

In further embodiments of the present technology, the connection orattachment locations can be a latch or latch system. A latch may be anymechanical fastener that is used to join two or more objects or surfacestogether while allowing for the regular separation of the objects orsurfaces so connected or attached. A latch may consist of a fastenerthat engages a catch, groove, hole, or suitable equivalent totemporarily affix or hold the base and the cover in close proximity.Suitable latching systems are known in the art. The latch may alsoconsist of at least one flexible singular piece of substrate (e.g., abiocompatible plastic) that engages with a hole (or alternatively overthe edge of) the base and/or cover to retain each so that they are inclose proximity, preferably in a temporary fashion or manner. Finally,the at least one connection or attachment location for coupledcommunication between the base and the cover may also include discretepoints or regions. In other embodiments, the at least one attachment orconnection location may be at least one side or a portion of a side ofthe base and cover. In some embodiments, the coupled communication maybe at least two sides (or portions of each side), alternatively at leastthree sides (or more) (or portions of such sides), among others.

Substrate Selection

In some aspects, the base 30 and/or the cover 60 can comprise anycompatible substrate for the practice of the present technology,preferably a biocompatible substrate. The substrate can providestructural integrity or support to the membrane 20 for handling duringany of the phases described herein (e.g., manufacture, storage,transport and/or final application). In some instances, the substratemay be a suitable composition that is not chemically or physicallyaltered by cryopreservation solutions (for example, solutions containingDimethyl Sulfoxide (DMSO). In some aspects, the substrate is also notchemically or physically altered by abrupt or large changes intemperature, and can be used within a wide temperature range (e.g.,−196° C. to 60° C.±5° C.). In other embodiments, the substrates are madeof polymers, copolymers, or biocompatible materials (e.g., plastics)that are thermal compatible and compatible with use withcryopreservation solutions. Suitable plastics include, but are notlimited to, low density polyethylene (LDPE), high density polyethylene(HDPE), ECTFE or ETFE copolymer (Ethylene ChloroTriFluoroEthylene orEthylene tetrafluoroethylene) FEP (fluorinated ethylene propylene), PE(Polyethylene) PP (Polypropylene), PMP (Polymethylpentene), Teflon®, PS(Polystyrene), RESMER™ (also known as RESMER Manufacturing Technologycommercially available from Thomas Scientific of Swedesboro, N.J.), EVA,among others. However, it should be appreciated by those skilled in theart that in those embodiments of the present technology in which acryopreservation media is not utilized, other suitable substrates areenvisaged in the practice of the present technology.

In further embodiments of the present technology, the base 30 of thepresent technology may comprise a material suitable for use withbiological or cellular materials, for example, a bio- orcellular-compatible plastic. In additional embodiments, the cover 60 maycomprise a material suitable for use with biological or cellularmaterials, for example, a bio- or cellular-compatible plastic. Theplastic may be a composite of different plastics or a homogenous plasticcomposition. Further such plastics may be combinations of plastics,layers of one or more types of plastics, among other plasticscombinations. Again, preferably, the plastics utilized in the practiceof the present technology are biocompatible plastics that are furtherpreferably, made of medical grade quality. It is also preferable thatthe substrates used in the practice of the present technology should becapable of withstanding a wide range of temperature changes ranging fromabout 40° C.±5° C. to about −196° C.±5° C., preferably from about 40°C.±5° C. to about −80° C.±5° C. The substrates should be capable ofwithstanding freezing temperature from about −80° C.±5° C. to about−196° C.±5° C. Substrates should also be capable of remaining at aboutroom temperature (about 20° C. to about 25° C.±5° C.), duringrefrigeration (about 4° C. to about 8° C.±5° C.), and during freezing(from about −20° C.±5° C. to about −196° C.+/−5° C.), alternatively fromabout −45° C. to about −50° C.±5° C., alternatively from about −80° C.to about −196° C.±5° C. For substrates used with cellular membranes, thesubstrates should be capable of withstanding a wide range oftemperatures from about 60° C. to about −196° C. (+5° C.). Suitablebiocompatible plastics may include, but are not limited to plastics thatwithstand exposure to a cryopreservation solution and/or membrane,tissue, graft or other biological material(s) without chemicalalteration of its composition and/or alternatively, damaging, injuring,or otherwise harming the membrane (tissue, graft, or other biologicalmaterial(s)), including harming viable cells associated therewith.Suitable biocompatible plastics can also include, for example, plasticscapable for use in 3-D printing applications or manufacturingprocedures.

Compositions, Devices, Articles of Manufacture and Systems

Some aspects of the present technology provide compositions, devices,articles of manufacture and systems (e.g., a membrane product package100) comprising a base 30, a cover 60, and a membrane 20 (tissue, graftor other biological material(s) temporarily associated with each. Again,the base 30 preferably comprises at least one membrane receiving portion40 wherein the membrane receiving portion temporarily contacts, supportsand holds at least one membrane 20 (tissue, graft, or other biologicalmaterial(s)). The membrane receiving portion 40 also preferably providessufficient surface traction to maintain the temporary adherence,attachment or connection of the membrane 20 (tissue, graft, or otherbiological material(s)) to the base 30 without curling of the edges ofthat membrane (tissue, graft, or other biological material(s)).

In other embodiments, and with reference to FIGS. 5A-7B, the membrane 20(tissue, graft, or other biological material(s)) is temporarily attachedvia at least one attachment or connection location associated with thebase 30. However, it should be appreciated by those skilled in the artthat the membrane 20 (tissue, graft, or other biological material(s))may be temporarily attached, connected, or adhered to the base by morethan one location, preferably two or more, three or more and the like.The locations, as described herein, may be discrete connection orattachment points 22 between the membrane 20 (tissue, graft, or otherbiological material(s)) and the base 30, including, but not limited to,cauterization points, points attached via a biocompatible adhesive, heatwelding, cauterization, ultrasonic welding, and other suitableattachment procedures for use with biological or cellular materials. Thenumber of locations 22 in which the membrane 20 (tissue, graft, or otherbiological material(s)) is temporarily attached to the base 30 issufficient to maintain, preferably, the planar orientation of themembrane (tissue, graft, or other biological material(s)) withoutcurling or rolling of the edges thereof on the base, and further toprovide a sufficient adhesion, attachment or connection such that themembrane (tissue, graft, or other biological materials(s)) preferablydoes not significantly move or slide during handling (e.g., whenattaching the cover, adding a cryopreservation medium, during storage,during transport, or removing the cover prior to end use application).

The temporary attachment or connection points 22 of the membrane 20(tissue, graft, or other biological material(s)) to the base 30 may alsobe in a pattern. For example, a suitable pattern for temporarilyattaching or connecting the membrane (tissue, graft, or other biologicalmaterial(s)) to the base can be at least one connection or attachmentpoint 22 in each corner of the base 30 (or membrane receiving portion 40thereof); at least one attachment point 22 in a middle portion of theupper edge (e.g., the edge farthest away from the handling portion, ifapplicable) of the base 30 (or membrane receiving portion 40 thereof),as viewed from the top, looking down at the base 30; and at least oneconnection or attachment point 22 in a middle portion of the lower edgeof the base 30 (or the lower edge of the membrane receiving portion 40thereof), as viewed from the top, looking down at the base 30. Othersuitable patterns are also envisaged, including the exemplary patternsdisplayed in Tables 7 and 8. Further, additional suitable patterns canalso include at least one attachment point in each corner of themembrane receiving portion 40 of the base and at least one attachmentpoint in a middle portion of the lower edge of the base. In someinstances the at least one attachment point comprises attachment pointsat the four corners of the membrane receiving portion; in someinstances, the attachment points are additionally in middle portions ofthe edges defined between the corners of the membrane receiving portion.In an exemplary embodiment, the membrane can be attached to the base atleast at the four corners and at least at one point in a middle portionof the upper and/or lower edge. The cover may be attached to themembrane and/or the base by at least one attachment point, preferably atleast two attachment points, more preferably at least at threeattachment points. The at least one attachment point may be a discretepoint in a middle portion along one or more edges of the membranereceiving portion and/or the cover. For example, the three attachmentpoints may be at two middle points along side edges of the membranereceiving portion and/or the cover and at one middle point along theupper edge of the membrane receiving portion.

The Membrane

As provided herein, it should be appreciated that the present technologycan be utilized for membranes, tissues, grafts, and other biologicalmaterials, collectively referred to herein as “membranes” 20. Thus, theterm “membrane” or “membranes” shall be used expansively throughout theinstant specification to encompass various cellular and/or biologicalmaterials suitable for use in the practice of the present technology. Itshould also be appreciated by those skilled in the art that “membrane”or “membranes” of the present technology can comprise natural“membranes”, synthetic “membranes” or combinations or derivativesthereof. For example, natural membranes can include but are not limitedto grafts, naturally derived membranes, and bioengineered membranescomprising living cells, further including, but not limited to,placental membranes, skin grafts, in vitro cultured grafts, tendongrafts, among others. Natural membrane may include allografts,autografts or xenografts. Natural membranes may be derived from mammals,including, for example humans. Bioengineered membranes can includeliving cells, extracellular matrix, biomolecules, at least one type ofcytokine, and combinations or derivatives thereof. Based on thestructure of the bioengineered membranes, other suitable materials orbiomolecules may be associated with the membrane. Other natural membranemay include, for example, at least one natural fiber, for example,silks. Further non-natural or synthetic membranes, for example, caninclude but are not limited to membranes containing at least onesynthetic fiber or compound, such as nylon, copolymers, polymers,including, but not limited to, PVA (polyvinyl acetate), PLA, (polyacticacid), PGA (polyglycolic acid), PCL (polycaprolactone), PLGA(poly(lactic-co-glycolic) acid), and the like. Further, it should beappreciated that suitable membranes can also include chorionic membraneproducts, amniotic membrane products, combinations thereof and otherplacental membrane products. Placental membranes products that can beused with the present technology are disclosed in U.S. application Ser.No. 13/030,507 (Publication No. 2011/0212158); Ser. No. 14/069,894(Publication No. 2014/0140966); Ser. No. 14/070,035 (Publication No.2014/0127317); Ser. No. 14/172,940 (Publication No. 2014/0294777); Ser.No. 14/056,101 (filed Oct. 17, 2013); Ser. No. 14/070,040 (PublicationNo. 2014/0127177); Ser. No. 14/272,343 (Publication No. 2015/0010609);and Ser. No. 14/291,256 (Publication No. 2014/0301986), in the name ofOsiris Therapeutics, Inc. of Baltimore, Md., all of which areincorporated by reference in their entireties. Suitable amnioticmembrane product includes Grafix® Prime® (Osiris Therapeutics, Columbia,Md.). Suitable chorionic membrane products include, for example, Grafix®Core® (Osiris Therapeutics, Columbia, Md.).

Other suitable grafts for use in the practice of the present technologycan also include, for example, grafts containing viable cells. Somesuitable grafts, for example contain fibroblasts, epithelial cells, stemcells, mesenchymal stem cells, and compositions comprising variouscombinations thereof. In some embodiments, the compositions comprisingviable fibroblast and epithelial cells.

Suitable bioengineered grafts include grafts in which viable cells, forexample fibroblasts, stem cells, epithelial cells, mesenchymal stemcells, which are seeded onto a synthetic or natural membrane. The cellsare cultured to provide a sufficient membrane structure. Based on thestructure of the bioengineered membranes, other suitable materials orbiomolecules may be associated with the membrane. For example,bioengineered grafts may contain extracellular matrix, biomoleculesincluding, but not limited to, cytokines, growth factors, co-stimulatorymolecules, proteoglycans, and the like. In some instances, thebioengineered grafts may not include viable cells, and may include otherbiological membrane components, including, but not limited to,extracellular matrix (e.g., collagen, proteoglycans), biomolecules andthe like.

As can be illustrated by the present technology it was surprisinglyfound that, the described compositions, devices, articles of manufactureand/or systems (e.g., the disclosed support assembly and membraneproduct package 100) maintain the viability of the temporarily attached,connected, or adhered cells in the membranes. Such an outcome isadvantageous as the present technology provides various packaging orpackaging system embodiments that support, protect, contain and maintainlive cells (e.g., naturally derived membranes or bioengineeredmembranes) for use in a variety of therapeutic, diagnostic, experimentaland/or analytical applications/uses unlike the conventional packaging,packaging systems and non-living cellular packaging products of theprior art.

Further, the compositions, devices, and systems of the presenttechnology also were surprisingly found to maintain the viability,reduce or prevent injury or damage and maintain an ease of removal andapplication of the cells in the temporarily attached membranes evenduring a variety of environmental stresses such as manufacturing,processing, cryopreservation, freezing, storage, thawing, transporting,and final application/use of such membranes (or cells). Such outcomesare advantageous for the support, stability and protection of cellularor biological products in a packaging or packaging system not envisagedby the conventional art.

Further, the compositions, devices, articles of manufacture and systemsof the present technology may also maintain the integrity of themembrane during a variety of environmental stresses, such as,manufacturing, processing, cryopreservation, freezing, storage, thawing,transporting, and final application/use of such membranes (or cells).

The membrane 20 is preferably placed on the base 30 in an operativeposition, which maintains the directionality of the membrane (e.g.,epithelial cells or tissues on the cover and connective tissue cells ortissue on the base). As described, the base and/or the cover may belabeled to maintain the directionality with a marker or label.Maintaining directionality is important in cellular repair, especiallywhen membranes which mimic the composition of the skin are used. Forexample, some membranes may have a first side (e.g., lower surface 28)and a second side (e.g., upper surface 26), where the first and secondsides have different compositions. For example, amniotic membranesderived from placental tissue have a first side (e.g., lower surface 28)containing stromal cells and a second side (e.g., upper surface 26)containing epithelial cells. For application as a wound or tissue defectrepair composition, it is important to maintain the directionality ofthe membrane. The first side containing stromal cells should make directcontact with the tissue defect or wound, and the epithelial layer shouldface exterior to the wound, mimicking the structure of the epidermis.

Membranes utilized in the practice of the present technology may be anysuitable size and customizable depending on the type of membrane and theparticular end application or usage of that membrane. Suitable sizes(length×width) of membrane include, but are not limited to, about 1.5cm× about 1.5 cm, about 2 cm× about 2 cm, about 3 cm× about 3 cm, about4 cm× about 4 cm, about 5 cm× about 5 cm, about 6 cm× about 6 cm, about7 cm× about 7 cm, about 8 cm× about 8 cm, about 7.5 cm× about 15 cm,about 1.5 cm× about 2 cm, about 1.5 cm× about 3 cm, about 2 cm× about 3cm, about 3 cm× about 4 cm, about 2 cm× about 5 cm, about 3 cm× about 5cm, about 4 cm× about 5 cm, about 5 cm× about 7 cm, about 5 cm× about 10cm, about 5 cm× about 15 cm, and include any variations or sizes andranges there between, in increment of 0.1 cm to 1 cm.

The compositions (e.g., kits) may further comprise a container. Thecontainer may be used to store the membrane-containing device orcomposition (e.g., the membrane product package 100). Additionally, thecontainer may be used for cryopreservation, for handling and shipping ofthe membrane. The container allows for the addition of cryopreservationsolution to the membrane. The container may be a bag or receptacle, orother suitable container. The container is made from a material which isable to be sterilized, can withstand cryopreservation solution and alsoa wide range of temperatures and/or freeze/thaw cycles without becomingbrittle or loosing integrity. Suitable containers include plasticcryopreservation bags, including, for example OSRSFP-90 CryogenicStorage bag.

In some embodiments, the composition (e.g., kit) further comprisescryopreservation solution. The cryopreservation solution is added to thecontainer containing the membrane-mounted device or composition.Preferably, a sufficient amount of cryopreservation solution is added tothe container to protect the membrane during the subsequent freezingsteps. The base containing the membrane receiving portion allows forsufficient infusion of the membrane with the cryopreservation solutionto maintain viability of the cells contained within the membrane.Suitable cryopreservation solutions are known in the art. In oneembodiment, the cryopreservation comprises storage in a cryopreservationmedium comprising one or more cell-permeating cryopreservatives, one ormore non-cell permeating cryopreservatives, or a combination thereof.Suitable cryopreservatives include, but are not limited to, DMSO, aglycerol, a glycol, a propylene glycol, an ethylene glycol, propanediol,polyethylene glycol (PEG), 1,2-propanediol (PROH) or a combinationthereof. In some embodiments, the cryopreservation solution may containone or more non-cell permeating cryopreservative selected from polyvinylpyrrolidione, a hydroxyethyl starch, a polysaccharide, a monosaccharide,an alginate, trehalose, raffinose, dextran, human serum albumin, ficoll,lipoproteins, polyvinyl pyrrolidone, hydroxyethyl starch, autologousplasma or a combination thereof. Other examples of usefulcryopreservatives are described in Cryopreservation (BioFiles, Volume 5,Number 4 Sigma-Aldrich® Datasheet).

For example, a suitable cryopreservation solution comprises acryopreservative, in an amount of at least about 0.001% to 100%,suitably in an amount from about 2% to about 20%, preferably about 5% toabout 10% by volume. In some instances, the cryopreservation solutioncomprises at least about 2% cryopreservative. Further, thecryopreservation solution may comprise serum albumin or other suitableproteins. In some embodiments, the cryopreservation solution comprisesfrom about 1% to about 20% serum albumin or other suitable proteins,alternatively from about 1% to about 10%. Serum albumin or othersuitable proteins are present to help stabilize the membrane during thefreeze-thaw process and to reduce the damage to cells, maintainingviability. Serum albumin may be human serum albumin or bovine serumalbumin. The cryopreservation solution may further comprise aphysiological buffer or saline, for example, phosphate buffer saline.

The container is filled with sufficient amount of the cryopreservationsolution to cover both sides of the membrane. The amount of thecryopreservation solution necessary will depend on the type of containerused and the size of the container relative to the size of themembrane-containing composition or device (e.g., membrane productpackage 100). The lower the amount of cryopreservation solutionnecessary to cover the composition/device, the faster the composition isable to thaw. Thus, it is desirable to use the least amount ofcryopreservation solution that allows for top coverage of the membranewithout compromising viability of the cells during the freeze thaw.Further, the smaller the membrane and the smaller the container used,the less cryopreservation solution can be used.

In some embodiments, a bag is used containing cryopreservation solutionin an amount from about 7 ml to about 50 ml, alternatively from about 10ml to about 50 ml, alternatively from about 15 ml to about 50 ml,alternatively from about 15 ml to about 25 ml. In one preferredembodiment, about 15 ml of cryopreservation solution is added to thecontainer or bag. The amount of cryopreservation solution can besufficient to fully submerge the membrane. The amount will depend on thesize of the bag used and the size of the membrane being cryopreserved.If a small bag is being used with a small (e.g. smaller than 2 cm×2 cmmembrane), about 3 ml to about 10 ml, alternatively 3 ml to about 7 mlof cryopreservation solution may be used.

In some embodiments a container is used containing from about 7 ml toabout 50 ml, alternatively from about 5 ml to about 20 ml, alternativelyfrom about 7 ml to about 20 ml, alternatively from about 7 ml to about15 ml. The amount of cryopreservation solution can be sufficient tofully submerge the membrane within the container. The amount will dependon the size of the container used and the size of the membrane beingcryopreserved.

In some embodiments, the amount of cryopreservation solution issufficient to protect cells during the freezing and subsequent thawingprocedures. In some embodiments, at least 70% cell viability ismaintained after a freeze-thaw. In some aspects, at least 75% cellviability is maintained, alternatively about 80% cell viability ismaintained, alternatively 85% cell viability is maintained,alternatively about 90% cell viability is maintained, alternativelyabout 95% cell viability is maintained. In some embodiments, atviability of the membrane is at least 70%, at least 71%, at least 72%,at least 73%, at least 74%, at least 75%, at least 75%, at least 78%, atleast 80%, at least 82%, at least 85%, at least 88%, at least 89%, atleast 90%, at least 92%, and percentages in between.

In some embodiments, the amount of cryopreservation solution issufficient to protect the structural, architectural, and or 3-Dstructure of the membrane, including a cellular matrixes. In someembodiments, the cryopreservation solution contains a cryopreservativein an amount of 0.01% to about 100%, alternatively from about 2% toabout 100%. In some embodiments, the cryopreservation solution containspolysaccharides or monosaccharides.

Methods of Using, Employing, or Applying the Compositions, Devices,Articles of Manufacture, or Systems

In various embodiments of the present technology, a method of making acryopreserved packaging product, cryopreserved cellular packagingproduct, or cryopreserved therapeutic packaged product (e.g., a membraneproduct package 100) is provided, among others. The methods comprise,for example, the steps of providing at least one membrane to becryopreserved; adhering, connecting or attaching, preferablytemporarily, the membrane to at least one membrane receiving portion ofa base; adhering at least one cover to the membrane and base; placingthe then membrane-containing base and cover into a storage receptacle;filling the storage receptacle with a sufficient amount of at least onecryopreservation media (e.g., a cryopreservation solution) to submerge,immerse, or permeate (partially or completely) the membrane containingbase and cover combination; placing the storage receptacle within arefrigerated environment at about 2° C. to about 8° C. for at leastabout 10 minutes, preferably about 30 to about 60 minutes; and thensubsequently subjecting the membrane containing base and covercombination to a freezing environment, thus freezing the container topreferably about −80° C. The cryopreserved product (i.e., the membranecontaining base and cover combination of the present technology) shouldremain frozen until thawed for use. In other embodiments, the method ofadhering, connecting or attaching the membrane to the base comprisescauterizing the membrane to the base at least at one point,alternatively at least at three points, alternatively at least at fivepoints, alternatively at least at six points. Further, the method ofadhering the membrane to the base could comprise using a biocompatibleadhesive to adhere at least one point between the membrane and the base,preferably at least five points, alternatively at least six points. Inother embodiments, the cover is adhered, connected or attached to themembrane-containing base by cauterizing at least at one point,alternatively at least at three points. Further, the adhering can bedone by using an adhesive at least at one point between the cover andthe membrane-containing base, at least at two points, at least at threepoints.

In some methodology embodiments of the present technology, a method ofcryopreserving a membrane is provided comprising the steps of: a)preparing a membrane; b) adhering the membrane to the device (orcomposition, article of manufacture, or system of the presenttechnology) as described herein; c) placing the device comprising themembrane in a sterile receptacle; d) applying a cryopreservation media(e.g., a cryopreservation solution) to the device in the receptacle,wherein the device comprising the membrane is partially or completelysubmersed, immersed, or is permeated in the cryopreservation media, ande) cryopreserving the sterile holder and device (i.e., containing theselected membrane) at a temperature of about −80° C. In otherembodiments, the method of cryopreserving a membrane may comprise thesteps of: preparing a composition (or device, article of manufacture, orsystem of the present technology) as described herein; placing thecomposition in a sterile receptacle; applying a cryopreservation media(again, e.g., a cryopreservation solution) to the device in thereceptacle, wherein the device (i.e., further comprising the membrane)is submerged, immersed, permeated partially or fully in thecryopreservation media; and cryopreserving the sterile receptacle anddevice at a temperature of about −80° C.±5° C. to about −196° C.±5° C.

Kits

Packaging kits comprising the compositions, devices, articles ofmanufacture, devices and systems of the present technology are alsoprovided. The kits can comprise, for example, a device comprising a basecontaining a membrane receiving portion and a cover with or without thetemporarily attached membrane as described herein. The kits can furthercomprise a device (composition, article of manufacture, or system) ofthe present technology and instructions for adhering, attaching orconnecting, preferably temporarily, the membrane as described herein to,preferably between, the base and the cover in some manner or fashionthat meets the goals and advantageous of the present technology asdescribed herein. In other aspects, the kits can provide furtherinstructions on maintaining the directionality of the membrane whenapplied, attached, adhered or connected to the cover and/or base as wellwhen stored, transported, handled and finally applied to a wound ortissue defect, or when used in another application. For example, suchinstructions can provide placing a first side of the membrane facing thebase and a second side facing the cover. Thus, the directionality of themembrane can be maintained, for example, during storage and application.The kit may further comprise at least one adhesive, such as at least onebio-adhesive. Thus, in some aspects, the kits of the present technologyprovide instructions for preferably temporarily adhering, attaching, orconnecting the membrane to the base at least at one point, alternativelyadhering, connecting, or attaching (preferably temporarily) the membraneat least at three points to the base, alternatively adhering, connectingor attaching (preferably temporarily) the membrane at least at fivepoints to the base, alternatively adhering, attaching or connecting(preferably temporarily) the membrane at least at six points to thebase. In other respective aspects, the instructions of the presenttechnology can provide instructions as to how to cauterize the membraneat varying points to the base. In other aspects, the instructions of thepresent technology can provide a method of using a bio-adhesive toadhere, attach, or connect (again, preferably temporarily) the membraneto the base at specific points by applying the bio-sealer to at leastone point between the membrane and base. In some aspects, the kitcomprises instructions on adhering, connecting or attaching the cover tothe base, wherein the membrane is located between the cover and thebase. In some embodiments, the cover is adhered, connected or attachedto the membrane at least at one point, preferably at least at threepoints. In such embodiments, the adherence may be to the membrane itselfor pass through the membrane to the base. The points of attachment maybe discrete points along the edge, over the partial or whole length ofone or edges, or over the entire surface area.

Kits of the present technology for treating a tissue defect are alsoprovided herein. The kits may comprise a composition (or device, orarticle of manufacture, or system) comprising at least one membrane, abase and at least one cover as described herein. The kit may alsoprovide for a cryopreserved membrane as described herein. The kit canalso comprise at least one set of instructions for thawing the membrane.The kit may further comprise instructions, for example, for rinsing orwashing the membrane (e.g., once thawed if previously cryopreserved) andinstructions for applying (e.g., in an oriented manner or position) themembrane to at least one wound or at least one tissue defect of apatient (human or animal). Such kits of the present technology may alsoinclude at least one preparation guide for preparing a tissue suitablefor use in accordance with the practice of the present technology. Suchkits may include, for example, an application guide as to how to applythe membrane to a wound or tissue defect. Alternatively, the applicationguide may comprise, for example, instructions on how to remove the coverfrom the membrane-bound base and methods of how to slide the membranefrom the base onto a suitable treatment site or for other use. Further,such kits may contain a sizing chart and instructions for sizing themembrane to a preferable size depending on the application or treatmentsite size. Such an outcome of the present technology allows for a healthcare provider to customize or tailor the membrane selected prior to orduring the resultant procedure with the patient. Such real time oron-demand capability has been surprisingly found by use of the presenttechnology and overcome several limitations of the prior art in whichfixed membrane products and product sizes do not allow for suchcustomization, much less on a real time or on-demand basis.

In still further embodiments, the kits of the present technology furthercomprise a cutting device, for example scissors or a scalpel. In otherembodiments, the kit may contain a buffer or thawing medium as well asother media necessary for the particular use or application of themembrane contained therein. In other embodiments, the kits of thepresent technology may contain, for example, forceps, tweezers, andother handling media. In some embodiments, the kit further comprises acontainer.

Methods Concerning the Compositions, Devices, Articles of Manufactureand Systems of the Present Invention

The methods, compositions, devices, articles of manufacture and systemsof the present technology described herein provide for a “membrane” asset forth herein which maintains or provides at least about 70% viablecells for application to an end user (e.g., to a wound or a tissuedefect) or for other diagnostic, experimental, or analytical uses. Ithas been surprisingly found that the methods, compositions, devices,articles of manufacture and systems of the present technology allow forthe support and protection of living or fresh cellular materials forlater use post-manufacture, storage, preservation, transport andhandling prior to final application or use unlike that of the prior art.Moreover, significant therapeutic advantages can be achieved by themethods, compositions, articles of manufacture, devices and systems ofthe present technology due to the significantly enhanced and maintainedcellular viability. Further, costs are reduced, and treatment modalitiesare streamlined or enhanced, all while providing a convenient and easeto use approach for a handler of the presently described packagingand/or packaging products.

Methods of applying a membrane to a patient (human or animal) in needthereof are provided with respect to the present technology. At leastone method comprises the steps of (i) obtaining a device, composition ofmatter, article of manufacture, or system of the present technologydescribed herein containing a membrane or biological compositiondescribed herein which has been cryopreserved and stored at about −80°C. to about −196° C. The method can further comprise the steps of (ii)thawing the membrane; (iii) optionally, rinsing or washing the membranewith a sterile physiological solution or other suitable biologicalmedium; (iv) removing the cover from the membrane and base; (v) andapplying the membrane from the base onto the patient as a unitaryoutcome or step. Optionally, the method can also comprise the steps of(i) assessing the orientation of the membrane as to the cover and thebase to retain the orientation (or directionality) of the membrane priorto application; and (ii) applying the membrane with the properorientation as provided by the composition, device, articles ofmanufacture, or system of the present technology. It should beappreciated by those skilled in the art and as further described hereinthat the orientation can be provided in a variety of manners includingan optional directional marker or label separate from or inclusive ofthe cover, the base, or both the cover and the base.

In other embodiments, a method of applying a membrane to a patient(human or animal) in need thereof comprises the steps of: 1) thawing areceptacle (e.g., a bag, or other suitable receptacle for biological orcellular materials) containing the cryopreserved product of the presenttechnology to about room temperature; 2) removing the cryopreservedproduct from the receptacle; 3) rinsing or washing the cryopreservedproduct in a physiological buffer, physiological medium, or othersuitable biological or cellular medium; 4) removing the cover from thebase and membrane; and 5) sliding the membrane from the base in asingular step and applying the membrane onto the area to be treated onthe patient. The step of sliding the membrane from the base may includethe further steps of breaking the temporary adherence, connection orattachment between the membrane and the base at the at least oneconnection or attachment location.

It has been surprisingly found that the compositions, devices, articlesof manufacture, systems and methods of the present technology providefor the membrane to be slid from the base in a manner which allows thehandler (e.g., a health care provider) to maintain the directionality ofthe membrane when applied to the affected area or location withoutcausing the membrane to curl, fold over onto itself, or otherwise besignificantly injured or damaged during the application process. Such anability, provides for additional therapeutic outcomes of the presenttechnology in that a greater portion of the treatment area receives themembrane; a greater amount of the viable cellular material of themembrane is applied to the treatment area; potentially fewerapplications of the membrane need to be utilized to the patient (thusleading to potentially improved treatment times); increased patientcompliance; increased health care provider receptivity to the use ofsuch cellular products, reduced cost, and other advantageous outcomes.Further, because more of the end product of the present technology canbe preserved and applied, less waste occurs than conventional productsof a potential similar nature.

Moreover, because the end products of the present technology aresupplied in such a manner as described herein, the end user has theability to tailor the size and shape of the end product such that theresultant membrane to be applied to a particular affected area (or otherapplication) can be done in a customized manner and potentially in areal time, on demand manner as needed. This is significantly differentand improved over other conventional products of a similar naturecurrently available.

Finally, the end products and method of application of the presenttechnology also surprisingly allow and provide for increased handlingcomfort and increased safety within the medical discipline because theend product edges can be smoothed. In doing so, the end product of thepresent technology is easier to handle and does not puncture or cut thehandlers appendages. This is significant in the health care industrywhere blood borne disease transmission is to be prevented.

Further, the present technology surprisingly allows for a reduced thawtime. As demonstrated in the examples herein, the thaw time forcryopreserved membranes in the devices, compositions, articles ofmanufacture or systems of the present technology is 10 fold reduced ascompared to conventionally packaged membranes. This reduction in thawtime not only limits the time that the membrane is exposed tocryopreservative factors, but also provides much for convenience for theend user/health care provider that is applying the membrane to asubject. As demonstrated, that thaw time for packaged membrane of thepresent technology is about 3 to 4 minutes, as opposed to at least 30minutes for a conventionally packaged membrane. Also, in some instances,the reduction of time exposed to thawed cryopreservation solution alsocan lead to increased viability of the cells contained within themembrane.

It should also be appreciated by those skilled in the art that the“membranes” of the presently described technology utilized in thefurther methods of the present technology can be used for application toa number of different types of wounds or tissue defects. Tissue defectscan include, but not limited to, wounds, abrasions, lacerations,incisions, ulcers, corneal wounds, among others. Ulcers include, but arenot limited to, dermal ulcers. Optionally, the wound is a laceration,scrape, thermal or chemical burn, puncture, or wound caused by aprojectile. Optionally, the wound is an epidermal wound, skin wound,chronic wound, acute wound, external wound, internal wounds, orcongenital wound. Such wounds may be accidental or deliberate, e.g.,wounds caused during or as an adjunct to a surgical procedure.Optionally, the wound may be closed surgically prior to administration.

The membranes disclosed herein are also useful in treating a number ofwounds including: tendon repair, cartilage repair (e.g. femoral condyle,tibial plateau), ACL (anterior crucial ligament) replacement at thetunnel/bone interface, dental tissue augmentation, fistulas (e.g.,Crohn's disease, jejunal-tube-based, tracheoesophageal), missing tissueat adhesion barriers (e.g. nasal septum repair, vaginal wall repair,abdominal wall repair, tumor resection), dermal wounds (e.g. partialthickness burns, toxic epidermal necrolysis, epidermolysis bullosa,pyoderma gangrenosum, ulcers e.g. diabetic ulcers (e.g. foot), venousleg ulcers), surgical wounds, hernia repair, tendon repair, bladderrepair, periosteum replacement, keloids, organ lacerations, epithelialdefects, and repair or replacement of a tympanic membrane.

For the variety of methods of application of the membrane via thepresent technology, it is preferable that the application be via topicaladministration. Alternatively, it can be used during surgicalapplications.

In another embodiment, a membrane is administered to a subject totopically treat a burn. Optionally, the burn is a first-degree burn,second-degree burn (partial thickness burns), third degree burn (fullthickness burns), infection of burn wound, infection of excised andunexcised burn wound, loss of epithelium from a previously grafted orhealed burn, or burn wound impetigo.

In a still further embodiment, a membrane is topically administered byplacing the membrane directly over the skin of the subject, e.g., on thestratum corneum, on the site of the wound, so that the wound is covered,for example, using an adhesive tape.

It should be appreciated by those skilled in the art that other forms ofadministering or applying the present technology are also envisaged. Forexample, the membrane delivered by the device, composition, article ofmanufacture or system of the present technology may be administered asan implant, e.g., as a subcutaneous implant.

In yet further embodiment, a membrane is topically, cutaneously,subcutaneously, and the like administered to the epidermis to reducefeatures of aging skin or scarring. Such treatment is also usefullycombined with so-called cosmetic surgery (e.g. rhinoplasty,rhytidectomy, etc.).

In another embodiment, a membrane is topically administered to theepidermis to accelerate healing associated with a dermal ablationprocedure or a dermal abrasion procedure (e.g. including laser ablation,thermal ablation, electric ablation, deep dermal ablation, sub-dermalablation, fractional ablation, and microdermal abrasion).

Other pathologies that may be treated with present technology include,for example, traumatic wounds (e.g. civilian and military wounds),surgical scars and wounds, spinal fusions, spinal cord injury, avascularnecrosis, reconstructive surgeries, ablations, and ischemia.

A membrane prepared and applied according to the present technology canoptionally be used to reduce adhesion or fibrosis of a wound.Postoperative fibrosis is a natural consequence of all surgically-basedwound healing. By example, postoperative peridural adhesion results intethering, traction, and compression of the thecal sac and nerve roots,which cause a recurrence of hyperesthesia that typically manifests a fewmonths after laminectomy surgery. Repeated surgery for removal of scartissue is associated with poor outcome and increased risk of injurybecause of the difficulty of identifying neural structures that aresurrounded by scar tissue. Therefore, experimental and clinical studieshave primarily focused on preventing the adhesion of scar tissue to thedura matter and nerve roots. Spinal adhesions have been implicated as amajor contributing factor in failure of spine surgery. Fibrotic scartissue can cause compression and tethering of nerve roots, which can beassociated with recurrent pain and physical impairment.

In additional aspects and embodiments of the present technology, methodsof maintaining the directionality of a membrane during processing,storage, cryopreservation, or during application to a subject (human oranimal) are disclosed. The methods may comprise, for example, the stepsof preparing a membrane, wherein the membrane has a first a second side,wherein the first and second side comprise different compositions; andadhering the membrane to the device (composition, article of manufactureor system also of the present technology) disclosed herein, where themembrane is disposed partially or completely between the base and cover.The method can also comprise the step of adhering, attaching orconnecting the membrane to the cover and the base wherein the first sideof the membrane is facing and connects or attaches to the base and thesecond side of the membrane is facing and connects or attaches to thecover. Such a method may further comprise the step of labeling the baseand/or cover by a marker (e.g., a symbol) to indicate the orientation ordirectionality of application of the product, or alternatively thedirectionality of the membrane to be applied or utilized in a mannerwhere orientation is desired. In some instances, the base is marked fororientation and the membrane is attached via a predetermined orientationto the base.

In other embodiments, the methods of the present technology furthercomprise the step of determining the adherence or surface traction ofthe first side and the second side of the membrane to, for example, thecover and/or the base, and orientating the membrane such that the sideof the membrane with higher surface traction is facing the base. In someembodiments, the membrane comprises a first side comprising stromalcells and a second side comprising endothelial cells, wherein the firstside is orientated to face the base. In other embodiments, the firstside of the membrane has a greater adherence to the cover.

In the preceding paragraphs, use of the singular may include the pluralexcept where specifically indicated. As used herein, the words “a,”“an,” and “the” mean “one or more,” unless otherwise specified. Inaddition, where aspects of the present technology are described withreference to lists of alternatives, the technology includes anyindividual member or subgroup of the list of alternatives and anycombinations of one or more thereof. Moreover, the disclosures of allpatents and publications, including published patent applications, arehereby incorporated by reference in their entireties to the same extentas if each patent and publication were specifically and individuallyincorporated by reference.

It is to be understood that the scope of the present technology is notto be limited to the specific embodiments described above. The presenttechnology may be practiced other than as particularly described andstill be within the scope of the accompanying claims. Likewise, thefollowing examples are presented in order to more fully illustrate thepresent technology. They should in no way be construed, however, aslimiting the broad scope of the technology disclosed herein.

The presently described technology and its advantages will be betterunderstood by reference to the following examples. These examples areprovided to describe non-exhaustive embodiments of the presenttechnology. By providing these examples, the scope of the presentlydescribed and claimed technology is not limited in spirit or scope. Itwill be understood by those skilled in the art that the full scope ofthe presently described technology encompasses at least the subjectmatter defined by the claims appending this specification, and anyalterations, modifications, derivatives, combinations, or equivalents ofthose claims. Further, the citations provided herein are herebyincorporated by reference for the cited subject matter.

EXAMPLES Example 1: Producing a Composition, Device, Article ofManufacture, or System According to the Present Technology

The following example demonstrates the use of a base and a cover of thepresent technology with a membrane, e.g. a placental product. Thefollowing procedure was used:

1. Placental membranes were prepared for placement in the packaging orhandling compositions, devices, articles of manufacture or systems ofthe present technology. Methods of preparing placental products, forexample, are disclosed in U.S. patent application Ser. No. 13/030,507(Publication No. 2011/0212158); Ser. No. 14/069,894 (Publication No.2014/0140966); Ser. No. 14/070,035 (Publication No. 2014/0127317); Ser.No. 14/172,940 (Publication No. 2014/0294777); Ser. No. 14/056,101(filed Oct. 17, 2013); Ser. No. 14/070,040 (Publication No.2014/0127177); Ser. No. 14/272,343 (Publication No. 2015/0010609); andSer. No. 14/291,256 (Publication No. 2014/0301986), which areincorporated herein by reference in their entireties.

2. In a pre-processing step, the membrane is prepared to size by anysuitable means. In this instance, the membrane was placed on temporarynitrocellulose paper substrate to allow for convenient handling andprovides a template for cutting to size.

3. Tissue was removed from the nitrocellulose paper using forceps andtransferred onto the perforated plastic base. For amniotic membrane, thestromal side is placed facing the perforations. The tissue was adjustedto cover the entire area of the membrane receiving portion of theplastic base. Any extra tissue was folded over the base.

4. The tissue was cauterized at the points indicated in FIGS. 7A-7B.FIGS. 7A-7B depict a schematic representation of cautery points on theplastic pieces (6 points on the base, left; 3 points on the cover,right). Side portions of a cautery pen were used to cauterize the fourcorners and the middle point of the cover. A tip portion of a cauterypen was used to cauterize the base middle point.

5. The cover was placed on top of the tissue and base, aligning thecorner edges.

6. The cover was cauterized to the membrane and base using the sideportions of the cautery pen at the three points shown in FIG. 7B.

Example 2: Cell Viability and Epithelial Growth Factor (EGF) Content ofMembranes

Samples were prepared as described in Example 1. After at least 48 hoursof deep freezing in a −80° C. freezer, batches of 8 samples were takenout and thawed in a room temperature water bath 18-24° C. and tested toensure minimum cell viability (>70%) and for the amniotic membrane, EGFcontents (>7.8 pg/ml). Results are shown in Tables 11-13 describedherein.

Tissue samples of appropriate size (5×5 cm or 2×2 cm) were used forvalidation assays. Refer to Table 1 for the number of membranes to betested per lot and the tests performed (Eastman Tritan MP100 Copolyester(Tritan™).

TABLE 1 Amniotic and Chorionic Membrane Testing Scheme Amniotic MembraneChorionic Membrane Test Method Nitrocellulose Tritan ™ NitrocelluloseTritan ™ Cell Viability Cell 1-5 cm × 5 cm 1-5 cm × 5 cm 1-5 cm × 5 cm1-5 cm × 5 cm count EGF ELISA 2-2 cm × 2 cm 2-2 cm × 2 cm N/A N/Aconcentration

Cell counts were performed. Briefly, amniotic membrane was digested withcollagenase and chorionic membrane was digested with collagenase andtrypsin for 15-45 min at 37° C. with constant agitation. Once tissue wascompletely digested or it reached the longest time point, the cellsuspension is either passed through a cell strainer and wash strainerwith Dulbecco's Modified Eagle Medium (DMEM) (chorionic membrane) orcentrifuged and the cell/tissue pellet resuspended in trypsin andincubated at 37° C. for another 5-15 min. After incubation the cellsuspension is passed through a cell strainer and wash strainer with DMEM(amniotic membrane). Centrifuge for another 10 min to obtain cellpellets. DMEM was added and cell counting performed using ahemocytometer with Trypan blue exclusion under a light microscope.Digested amniotic or chorionic samples were diluted to the appropriateratio in Trypan Blue and the number of cells in 5 x 0.0001 ml squareswere counted.

Human EGF ELISA assay was performed to measure the contents of human EGFof amniotic membrane. Briefly, two 2×2 cm tissues were used. Membranewas snap frozen in liquid nitrogen for 5 minutes. 500 μl of calibratordiluents was added and the tissue was homogenized in a Tissue Lyserchamber for 3 minutes at 50 Hz. Samples were centrifuged at 14,000 rpmfor 10 minutes at 4° C. The supernatant was used for ELISA assay. Theassay is described in details in manufacturer manual. Three lots ofhuman amniotic membrane were randomly selected and assayed.

Example 3: Evaluation Methods of Different Base Configurations

Mounted membranes were inspected for attachment with the packaging. Thepassing criteria included that 1) all components of the mountingapplicator (the support assembly) and tissue stay together; and 2) atleast 75% of the tissue should stay flat on the mounting applicator forall the following procedures:

Rinsing in Dulbecco's Modified Eagle Medium (DPBS)

Forceps were used to hold a corner of the base without touching themounted membrane. The membrane was rinsed in saline solution. Themembrane was monitored for detachment from the base.

Filling of the Cryoprotectant Solution

The membrane assembled within the base and cover (as in Example 1) wasplaced into an OSRSFP-90 Cryogenic Storage bag. The bag was filled with50 ml cryoprotectant solution. The membrane was monitored for detachmentfrom the plastic base, and the cover was monitored for detachment fromthe membrane and/or plastic base.

Rinsing Post-Thawed

After at least 18 hours of deep freezing at −80° C., amniotic membranewas thawed in sufficient volume of room temperature saline (such thatthe liquid surface could cover the entire Cryogenic Storage bag) untilall ice crystals disappeared. The membrane was monitored for detachmentfrom the plastic base during thawing. Once thawed, the membrane wasremoved from the OSRSFP-90 Cryogenic Storage bag and placed in the rinsebasin containing sterile saline. The membrane was monitored duringrinsing for detachment from the base and the cover was monitored fordetachment from the membrane and/or plastic base.

Application

The cover was removed and the tissue was observed to determine if morethan 75% of the tissue stayed attached to the base. The membrane waspromptly applied onto the wound such that its orientation was maintainedthroughout the process.

Example 4: Non-Nitrocellulose Approaches

Coated nitrocellulose was found to introduce chemicals of animal originsinto subjects, thereby rendering the coated nitrocellulose unsuitablefor some patients. Non-nitrocellulose approaches were thereforeevaluated.

For the prototypes developed under this category, plastics wereidentified as a material that provided for easy application in thatmembranes and tissues can slide off the plastic onto the wound bed whilemaintaining their orientation. This configuration included two plasticpieces generally identified as a “cover” and a “base”. The base was usedto maintain the orientation of the tissue, includes a portion having anon-uniform surface (a tissue/membrane receiving portion), and providesa platform for the sliding application. The cover provides protectionfor the tissue/membrane during manufacturing, transport, storage, anduse (e.g., thawing). The cover can minimize the fluid shear stressacting on the epithelial layer of the tissue (membrane) during thefilling of the cryopreservation solution as well as during the transitand handling of the cryogenic storage bags to/from a refrigerator into adeep freezer. Different chemical and mechanical fixation/attachmentmethods have been evaluated for the base and are summarized in Table 2(below). The perforation sandwich plastic design was selected forfurther development as it passed all evaluation criteria listed inExample 3

TABLE 2 Description of non-nitrocellulose paper prototypes and theirevaluation results. Prototype name Prototype description Poly-D-Lysin-Coat plastic with 1 mg/ml Poly-D- treated plastic Lysin solution for 3hrs at 37° C. Place amnion onto the coated plastic with stromal sidefacing plastic. Incubate at 37° C. for 3 hrs Sandpaper- Plastic weretreated with treated Plastic sandpaper. The entire surface feels roughto touch. Microplate Sandwich the tissue in between the Devices twoparts of a Microplate Uniseal, UniSeal ™ which contain anadhesive-backed clear polyetyrene seal film and a water-resistant paper.4 pins Fix four corners of tissue samples with pins so it stretches outto a square shape 4 straight slit Cut four corners of the base piececorners into slits, then insert each corner of the tissue inside theslits 4 straight slit Cut four corners of the base into corners withhard slits; align tissue onto a piece of paper inserts thick paper(blue) that was cut to the size of the units, i.e. 5 × 5 (cm²) or 2 × 2(cm²); then tuck the paper with the tissue inside the slits Two straightslits Cut two angled slits on the base on the base with sheet to anchorthe tissue. Cut two cover piece corners parallel to the first two slitssealed together on the plastic cover. Seal two with base plastic sheetstogether shown as the grey line. Place the tissue on the base sheet andtuck two base corners inside the two slits 4 clipping round Create fourcorners of the base corners piece to half-moon shape using a 3 mm biopsypunch; then tuck tissue in; tighten the tissue by pressing the back sideof the opening until hearing a click sound Frame with Make a paper framethe same size plastic lamination as the units, i.e. 5 × 5 (cm²) or 2 × 2(cm²); lay it on the top of one piece of plastic; then insert tissuesamples inside the frame; put another piece of same size plastic toenclose everything; laminate the four sides Clamp The tissue membrane isfastened in between two parts of the clamp and fastened by the screwPlastic Holes (5 mm in diameter) were “Sandwich” with punched using acomb hole perforations puncher paper binder binding machine

Plastic Selection

Based on general performance of the above described embodiments, theplastic “sandwich” configuration was selected for further evaluation.The next step was to identify plastic that: is compatible with livingtissue, low temperature, and DMSO; and complies with USP class VItesting (for plastics)

Copolyesters and polycarbonates were considered as potential materialsfor development. Tritan™ is described in the examples below as it wasprovided in a convenient validation package.

Example 5: Testing of Perforated Sandwich Design on Tritan™ SheetsPerforation Parameters

Sizes of the perforations and center-to-center spacing between adjacentperforations were varied (Table 3). Four combinations of perforationsize and center-to-center spacing were chosen as listed in Table 3.Placental membranes, either amniotic or chorionic membrane, were mountedon perforated plastic and evaluated according to performance criteriadescribed in Example 3 including: Rinsing in DPBS; Filling of theCryoprotectant solution; Rinsing post-thawed; and Application.

TABLE 3 Perforation parameters testing scheme PerforationCenter-to-Center Diameter Spacing 4 mm 10 mm 2 mm 10 mm 2 mm  5 mm 1 mm 4 mm

Neiko Hand Held Power Punch kit or a 1 mm biopsy punch was used tomanually create the perforations. AutoCAD file was generated and anevenly distributed perforation design was printed onto the Tritan™ sheetfor easy tracing. 2 mm and 4 mm were selected because perforationconfiguration with 5 mm holes was not able to pass the visual inspectioncriteria listed. The schematic representations of the perforation designand results are summarized in FIG. 10.

The results show placental tissue mounted on Tritan™ sheet with 1 mmperforation spaced 4 mm apart meet all criteria listed. Therefore theseperforation size and spacing parameters were selected for further cellviability and EGF testing.

Example 6: Evaluation of Different Perforation Patterns on the Tritan™Sheet

Because the high number of holes potentially leads to highermanufacturing cost, we sought to test whether different perforationpattern design would allow us to reduce the number of holes needed toprovide strong bonding (maintain shape and attachment).

1 mm diameter was used and different perforation patterns were tested asdescribed in FIG. 11. AutoCAD files of the pattern were generated andprinted onto Tritan™ sheets. Holes were created using 1 mm biopsypunches according to the printed design. Amniotic membrane was thenmounted onto the plastic. Top solid plastic pieces were used to coveramniotic membrane mounted on the perforated plastic. The evaluation ofeach pattern was done based on the criteria listed above. The evaluationof each pattern was done based on the criteria described in Example 3,including: Rinsing in DPBS; Filling of the Cryoprotectant solution;Rinsing post-thawed; and Application.

Results are summarized in FIG. 11. None of the described designsprovided results that were as effective as the most effective patternsin FIG. 10.

Results are summarized in Table 5. None of the described designsprovided results that were as effective as the most effective patternsin Table 4.

Example 7: Evaluation of Perforations on the Front or Back or BothPieces of the Plastic

Different combinations of the perforated and solid plastic pieces weretested. In our hands the combination of the solid plastic cover and theperforated plastic base allows the good attachment of amniotic membranewith easy removal of the cover without lifting/detaching amnioticmembrane from the base. Results are summarized in Table 6.

TABLE 6 Comparison between different perforation positions ConfigurationComments Perforation on the amniotic membrane tends to cover only adhereto the cover Perforations on Could not consistently keep both cover andmore than 75% membrane on base the base Perforations on The cover waseasy to be the base only removed amniotic membrane attaches well to theplastic base

Example 8: Different Cautery Systems and Cautery Patterns

The improved two-piece packaging system embodiments are suitably heldtogether with the tissue to provide as one unit to the customers. Herewe investigated different cautery systems and cautery patterns as thefixing method so that the chosen cautery system and pattern 1) caneffectively fix the tissues as well as the two plastic pieces togetherwithout breaking apart; 2) avoid burned brown spots of the tissue; and3) is cost-effective and can be used in a cleanroom environment.

Cautery Systems

Different cautery systems were evaluated as seen in Table 6. The maximumnumber of cautery points for each system was recorded. The performancecriteria were described in Example 3. The Bovine Fine Tip Cautery (704°C.) configuration and the Bovine Vasectomy Micro Fine Tip Cauteryconfiguration were found to provide the best performance and be the mosteconomical. The other configurations were found to have higher costs,provide fewer cautery points, produce more burned products, or have atendency to catch fire.

TABLE 6 Comparison Between Different Cautery Systems Number ofConfiguration Cauterizations Gemini Cautery Kit N/A Bovie Micro Fine Tip31~54 Cautery, 454° C. Accu-temp ½″ shaft  27 with fine tip, 677° C.Bovie Fine Tip  99~168 Cautery, 704° C. Bovie Vasectomy  90 Micro FineTip Cautery, 871° C. Bovie Vasectomy N/A Micro Fine Tip Cautery, 982° C.Accu-Temp Vasector N/A Fine Tip Cautery, 982° C. Bovie Micro Fine Tip464 Cautery, 1093° C. Bovie Fine Tip N/A Cautery, 816-1149° C. BovieFine Tip N/A Cautery, 1204° C.

Cautery Patterns

Different cautery patterns were evaluated for amniotic membrane as seenin FIG. 12 and chorionic membrane as seen in FIG. 13. The goal for theseembodiments was to use the least number of cautery points to meet allperformance criteria.

Design 1 (with eight points) was found to work best for amnioticmembrane, with five points on the base and three points on the cover.For chorionic, Design 1 was found to work the best, with six points onthe base and three points on the cover. In order to simplify themanufacturing process, it is contemplated that Design 1 for thechorionic membrane can be used to support both placental membranes.

For the amniotic membrane experiment, Designs 2-9 either did notconsistently meet the performance criteria or were found to have toomany cautery points (resulting in an overly complicated manufacturingprocess). Similarly, for the chorionic membrane experiment, Designs 2-9either did not consistently meet the performance criteria or were foundto have too many cautery points (resulting in an overly complicatedmanufacturing process). However, it is contemplated that Designs 2-9 forthe amniotic membrane experiment and Designs 2-9 for the chorionicmembrane experiment can be used as disclosed herein to support amembrane or other biological product.

Example 9: Effect of New Plastic Packaging on Placental Products

Results in Table 9-11 demonstrated that the plastic packaging has nonegative effect on cell viability for both membranes (amniotic andchorionic) and EGF content for amniotic membranes.

Effect of New Plastic Packaging on Cell Viability of Placental Membranes

TABLE 9 Evaluation of cell viability of amniotic membrane on Tritan ™Assay Donor Cell Acceptance Information Conditions Viability (>70%) 1Control 77% Pass Tritan ™ 70% Pass 2 Control 79% Pass Tritan ™ 73% Pass3 Control 71% Pass Tritan ™ 78% Pass 4 Control 78% Pass Tritan ™ 73%Pass 5 Control 94% Pass Tritan ™ 90% Pass 6 Control 87% Pass Tritan ™84% Pass

TABLE 10 Cell Viability of chorionic membrane Assay Donor CellAcceptance Information Conditions Viability (>70%) 7 Control 81% PassTritan ™ 87% Pass 8 Control 86% Pass Tritan ™ 79% Pass 9 Control 81%Pass Tritan ™ 89% Pass

Effect of New Plastic Packaging on EGF Content of Amniotic Membrane

TABLE 11 Evaluation of EGF contents of amniotic membrane on Tritan ™ byELISA EGF Assay Donor Concentration Acceptance Information Conditions(pg/ml) (>7.8 pg/ml) 10 Control 109.5 Pass Tritan ™ 157.3 Pass 11Control 33.3 Pass Tritan ™ 43.5 Pass 12 Control 95.6 Pass Tritan ™ 69.6Pass

Thus, as exemplified by the above-described illustrative embodiments, amounting applicator (support assembly) has been designed for membraneand tissues that can be derived from natural or non-natural (i.e.,synthetic) sources. The applicator (support assembly) can replacecurrent technology based on nitrocellulose paper and provides a simpleapplication procedure for the end user. As further disclosed herein, theapplicator (support assembly) can comprise a cover and a base, suitablyof a plastic material, that are positioned to accept a membrane betweenthe cover and base. The base has an irregular surface, such as aperforated portion, which receives a membrane, having a size generallycorresponding to its cover, and a tab area for handling purposes. Thebase can be sealed to the tissue (e.g., membrane to base) at a pluralityof points, and additional seal points can secure the cover to themembrane and/or base to make a “sandwich” configuration.

Example 10: Method of Thawing and Application of a CryopreservedMembrane Product of the Present Technology

Placental membrane products can be cryopreserved and stored frozen andshipped to the end user in a Styrofoam container.

Thawing:

Prior to use of the placental membrane product, two basins, a bottle ofsterile saline, scissors, sterile forceps and gloves can be gathered.One large basin can be used for thawing and a smaller sterile basin forrinsing.

The placental membrane product can be removed from storage. Theplacental product can be packaged in a carton box with tamper evidentlabels on each side of the box which describe product name and size, lotnumber, unit number, date of expiry, part number and required storageconditions. Inside the box, the product can be provided in achevron-type peel pouch, along with the package insert and chart labelsto be used on the patient's records.

The outer pouch can be peeled open and the inner cryobag can be placedinto the large thaw basin. To ensure the orientation of the placentalproduct is correct, the cryobag can be placed in the thaw basin with thelabel side up so it is visible to read. Sufficient warm water or salinecan be added into the large thaw basin containing the placental productto completely cover the cryobag. The water temperature should not exceedabout 39° C. or 102° F.

While the product is thawing, sterile saline can be added to the smallrinse basin. Once all ice crystals are completely thawed, the placentalproduct (graft) can be removed from the cryobag. The placental productshould remain in the thaw basin for no more than 15 minutes. Holding thecryobag with the port side down, the top of the bag can be cut withsterile scissors, taking care not to cut near the graft. With sterileforceps, the placental product (graft) can be removed from the cryobag.

In an operating room setting, sterile tools and basins should be used,and aseptic technique should be applied when thawing the placentalproduct.

Next, the placental product can be placed into the sterile rinse basinand confirm that the lettering on the base is oriented correctly (e.g.,the base correctly reads “PRIME” or “CORE” as shown in FIGS. 6A and 7A).This can allow for correct orientation for placement onto a wound, forexample, for amniotic membranes. The membrane should be applied to thesubject or patient within one hour.

Application:

The index wound identified for placental membrane placement should beappropriately cleaned and debrided.

To apply the placental product, the base can be held on the labeled tab,and the top plastic cover can be removed. Once the cover is off, theplacental product can be slid from the plastic backing onto the woundbed using aseptic technique.

Next, using sterile forceps, sterile gloves, or sterile moist cottonapplicators, the graft can be maneuvered to ensure that the entire woundbed is covered. The graft should be in direct contact with all surfacesof the wound bed, including the edges.

Excess membrane can be placed on the edges of the wound, can extend overthe surrounding healthy tissue, or can be folded into the wound bed. Anyair bubbles and pockets that may exist between the graft and the woundshould be removed for best results.

The placental product can optionally be covered with a non-adherentdressing, and an appropriate compressive or outer layer dressing can beapplied, depending on wound type.

Example 11: Comparison of Thawing Time for Placental Tissue Mounted onEither Disclosed Support Assembly (Tritan™) or Nitrocellulose Paper

Objective: To investigate whether there is a significant reduction ofthawing time when using the disclosed support assembly (base and cover),the current thawing procedures were performed on placental membranemounted on the disclosed support assembly and on nitrocellulose paper.

Methods:

Two samples were taken from two lots of placental tissue, with onesample of each lot packaged in the disclosed support assembly and theother sample of each log packaged on nitrocellulose paper. All sampleswere removed from the deep freezer (−80° C.).

A cryobag containing the placental tissue was positioned in the thawingbasin, which was filled with room temperature water.

Record Thawing Time

For placental tissue on nitrocellulose paper, the timer was stopped whenall ice crystals were not visible.

For placental tissue on the support assembly disclosed herein, the timerwas stopped when the plastic of the support assembly could be separatedfrom its surrounding ice crystals.

TABLE 12 Results: Thawing Time (min) Donor Nitrocellulose paper Tritan ™A 24 3 B 28 4

Because of the unique packaging design (plastic-membrane-plastic“sandwich”) of the disclosed support assembly, when thawing issufficient to loosen surrounding ice crystals, it is safe to take thesupport assembly out of the bag and simply remove the ice chunks oneither side of the plastic without damaging the membrane enclosed inbetween.

In contrast, for placental tissue mounted on nitrocellulose paper, thethawing had to be complete, i.e. all ice crystals disappear. Otherwise,the weight of the ice could tear the membrane or cause the membrane tofall off of the nitrocellulose paper, leading to self-folding whenthawing is complete.

Conclusion: The disclosed plastic packaging allowed a 8-10 times fasterthawing time than conventional nitrocellulose paper packaging.

Exemplary Devices, Methods, and Kits

In various exemplary aspects, disclosed herein is a support assembly forsupporting a biological product in an operative position, the supportassembly comprising: a base having a longitudinal axis and comprising aproduct receiving portion, the product receiving portion having a topsurface and an opposed bottom surface that are spaced apart relative toa vertical axis that is perpendicular to the longitudinal axis of thebase, wherein the product receiving portion comprises antraction-creating feature, wherein the traction-creating feature isselected from the group consisting of (i) a rough top surface and (ii) aplurality of perforations extending between the top and bottom surfacesof the product receiving portion; and a cover having a longitudinalaxis, a top surface, and an opposed bottom surface, wherein the cover isconfigured for releasable coupling to the base in a product-coveringposition, and wherein, in the product-covering position, the coveroverlies the product receiving portion of the base, wherein the base andthe cover are configured to cooperate to support the biological productin the operative position, wherein, in the operative position, thebiological product is positioned in engagement with at least a portionof the top surface of the product receiving portion of the base and atleast a portion of the bottom surface of the cover.

In another exemplary aspect, the base further comprises a handlingportion positioned adjacent to the product receiving portion relative tothe longitudinal axis of the base. In another exemplary aspect, in theproduct-covering position, the cover does not overlap with the handlingportion of the base. In another exemplary aspect, the handling portioncomprises a tab. In another exemplary aspect, the handling portion ofthe base has a longitudinal length and a width, wherein the productreceiving portion of the base has a longitudinal length and a width, andwherein the width of the product receiving portion is equal to the widthof the handling portion.

In another exemplary aspect, in the product-covering position, thelongitudinal axis of the cover is positioned in substantial alignmentwith the longitudinal axis of the base. In another exemplary aspect, theproduct receiving portion of the base has a longitudinal length and awidth, wherein the cover has a longitudinal length and a width, andwherein the longitudinal length of the cover is substantially equal tothe longitudinal length of the product receiving portion. In anotherexemplary aspect, the width of the cover is substantially equal to thewidth of the product receiving portion.

In another exemplary aspect, the cover has a plurality of corners, andat least one of the corners of the cover is rounded. In anotherexemplary aspect, the cover has four rounded corners. In anotherexemplary aspect, the product receiving portion of the base has tworounded corners, and wherein, in the product-covering position, tworounded corners of the cover overlie the two rounded corners of theproduct receiving portion of the base.

In another exemplary aspect, the traction-creating feature of theproduct receiving portion of the base comprises a plurality ofperforations. In another exemplary aspect, the plurality of perforationsof the product receiving portion of the base are substantially evenlydistributed throughout the product receiving portion. In anotherexemplary aspect, the plurality of perforations of the product receivingportion of the base are randomly distributed throughout the productreceiving portion. In another exemplary aspect, each perforation of theplurality of perforations has a respective diameter ranging from about0.1 mm to about 5 mm. In another exemplary aspect, each perforation ofthe plurality of perforations has a respective center point, and whereinthe center points of neighboring perforations are spaced apart by adistance ranging from about 0.35 mm to about 10 mm.

In another exemplary aspect, the traction-creating feature of theproduct receiving portion of the base comprises a rough top surface.

In further exemplary aspects, the disclosed support assembly can beprovided as part of a membrane product package, which further comprisesa membrane positioned in an operative position between the productreceiving portion of the base and the cover, wherein the membrane ispositioned in engagement with at least a portion of the top surface ofthe product receiving portion of the base and at least a portion of thebottom surface of the cover.

In another exemplary aspect, the membrane is attached to the top surfaceof the product receiving portion of the base at least one attachmentpoint. In another exemplary aspect, the membrane is attached to the topsurface of the product receiving portion of the base at least threeattachment points. In another exemplary aspect, the membrane is attachedto the top surface of the product receiving portion of the base at leastfive attachment points. In another exemplary aspect, the top surface ofthe product receiving portion of the base is attached to the cover atleast one attachment point.

In another exemplary aspect, the cover is attached to the membrane atleast one attachment point. In another exemplary aspect, the cover isattached to the membrane at least two attachment points. In anotherexemplary aspect, the cover is attached to the membrane at least threeattachment points. In another exemplary aspect, the top surface of theproduct receiving portion of the base is attached to the cover at leastone attachment point.

In another exemplary aspect, the top surface of the product receivingportion of the base is attached to the cover at least one attachmentpoint. In another exemplary aspect, the top surface of the productreceiving portion of the base is attached to the cover at at least threeattachment points.

In another exemplary aspect, the attachment points between the base andthe membrane, between the cover and the membrane, and/or between thebase and the cover are cauterization points.

In another exemplary aspect, the membrane is a natural membrane.

In another exemplary aspect, the membrane is a placental tissue product.In another exemplary aspect, the membrane is a chorionic membraneproduct. In another exemplary aspect, the membrane is an amnioticmembrane product.

In another exemplary aspect, the membrane is a synthetic membrane.

In another exemplary aspect, the membrane and the top surface of theproduct receiving portion of the base have sufficient surface tractionto maintain the membrane in the operative position following removal ofthe cover from the base.

In another exemplary aspect, the membrane and the top surface of theproduct receiving portion of the base have a first surface traction,wherein the membrane and the cover have a second surface traction, andwherein the second surface traction is lower than the first surfacetraction.

In additional exemplary aspects, the disclosed support assembly can beused in a method of producing a membrane product package, the methodcomprising positioning a membrane in an operative position between theproduct receiving portion of the base and the cover of the supportassembly, wherein the membrane is positioned in engagement with at leasta portion of the top surface of the product receiving portion of thebase and at least a portion of the bottom surface of the cover.

In another exemplary aspect, the step of positioning the membrane in theoperative position comprises: attaching the membrane to the top surfaceof the product receiving portion at a plurality of attachment points;and attaching the membrane to the cover at a plurality of attachmentspoints. In another exemplary aspect, when the traction-creating featureof the product receiving portion comprises a plurality of perforations,the method further comprises positioning the base, the membrane, and thecover within a cryopreservation solution, wherein the plurality ofperforations of the product receiving portion provide contact betweenthe membrane and the cryopreservation solution sufficient tocryopreserve the membrane.

In further exemplary aspects, a method of applying a membrane isprovided, comprising removing the cover from the disclosed membraneproduct package to expose a top surface of the membrane; disengaging themembrane from the top surface of the product receiving portion of thebase; and selectively applying the membrane to a desired location on ahuman or animal patient.

In still further exemplary aspects, the disclosed membrane productpackage can be provided as part of a kit for repairing a tissue defect.In another exemplary aspect, the kit can further comprise a containerthat encloses the membrane product package, wherein the container can beselectively opened to provide access to the membrane product package. Inanother exemplary aspect, the kit can further comprise instructions forapplying the membrane of the membrane product package to repair thetissue defect. In still further exemplary aspects, the kit can furthercomprise a cryopreservation solution. In still further exemplaryaspects, the kit can further comprise a basin configured to receive themembrane product package. In still further exemplary aspects, the kitcan further comprise scissors. In still further exemplary aspects, thekit can further comprise tweezers.

More generally, in further exemplary aspects, disclosed herein is adevice (e.g., a support assembly) comprising: a base comprising aproduct (e.g., membrane) receiving portion; and a cover; and at leastone location in which the base and the cover are in communication (atleast one temporary or removable attachment between the back and thecover.

In another exemplary aspect, the membrane receiving portion comprises atraction-creating feature (e.g., a structured surface). In anotherexemplary aspect, the traction-creating feature (e.g., structuredsurface) is selected from the group consisting of an abraded surface, arough surface, a scratched surface, a surface comprising a plurality ofperforations; a surface comprising a plurality of channels; a surfacecomprising a plurality of grooves; a surface comprising a plurality ofindentations.

In another exemplary aspect, the traction-creating feature (e.g.,structured surface) of the membrane receiving portion comprises asurface comprising a plurality of perforations. In another exemplaryaspect, the plurality of perforations are uniformly distributedthroughout the membrane receiving portion. In another exemplary aspect,the plurality of perforations are randomly distributed throughout themembrane receiving portion. In another exemplary aspects, the pluralityof perforations are evenly distributed in close relationship to eachother throughout the membrane receiving portion to provide sufficientsurface traction for the membrane to adhere to the base. In anotherexemplary aspect, the plurality of perforations are about 1 mm to about5 mm in diameter and each perforation is from 4 mm to about 10 mm apartas measured center to center. In another exemplary aspect, the pluralityof perforations are from about 0.1 mm to about 5 mm in diameter. Inanother exemplary aspect, the plurality of perforations are from about0.1 mm to about 1 mm in diameter. In another exemplary aspect, theperforations are spaced about 0.35 mm to about 10 mm apart as measuredcenter to center. In another exemplary aspect, the perforations arespaced about 4 mm to about 10 mm apart as measured center to center. Inanother aspect, the plurality of perforations are of a geometrical shapeor a nongeometrical shape. In another exemplary aspect, the plurality ofperforations are a shape selected from the group consisting of oval,rectangular, square, diamond, trapezoid, star, hexagonal, octagonal,semi-circular, crescent, or a combination thereof.

In another exemplary aspect, the traction-creating feature of themembrane receiving portion comprises a rough plastic surface.

In another exemplary aspect, the base comprises at least one plastic orpolymer.

In another exemplary aspect, the cover comprises at least one plastic orpolymer.

In another exemplary aspect, the plastic base further comprises ahandling portion adjacent to the membrane receiving portion.

In another exemplary aspect, the handling portion does not overlap withthe cover.

In another exemplary aspect, the handling portion comprises a tab. Inanother exemplary aspect, the tab spans the entire width of the base.

In another exemplary aspect, the cover spans the entire membranereceiving portion of the base.

In another exemplary aspect, the at least one location in which the baseand cover are in communication comprises at least one cauterizationpoint, at least one point made by an ultrasonic welder, or at least onepoint comprising an adhesive. In another exemplary aspect, the at leastone location in which the base and cover are in communication comprisesa plurality of points.

In another exemplary aspect, the base and the plastic cover are formedfrom a single piece of plastic.

In another exemplary aspect, the base and the plastic cover are separatepieces of plastic.

In another exemplary aspect, the base and the cover are made of the sameplastic.

In another exemplary aspect, base comprises plastic selected from thegroup consisting of polycarbonate, copolyester, low density polyethylene(LDPE), high density polyethylene (HDPE), ECTFE copolymer, ETFEcopolymer, FEP (fluorinated ethylene propylene), PE (Polyethylene), PP(Polypropylene), PMP (Polymethylpentene), Teflon, PS (Polystyrene), EVA,and Tritan copolyester MP100.

In another exemplary aspect, the plastic of the cover is selected fromthe group consisting of polycarbonate, copolyester, low densitypolyethylene (LDPE), high density polyethylene (HDPE), ECTFE copolymer,ETFE copolymer, FEP (fluorinated ethylene propylene), PE (Polyethylene),PP (Polypropylene), PMP (Polymethylpentene), Teflon, PS (Polystyrene),EVA, and Tritan copolyester MP100.

In another exemplary aspect, the cover is a solid sheet of plastic.

In another exemplary aspect, the cover has non-sharp corners. In anotherexemplary aspect, the corners are rounded.

In another exemplary aspect, the base has non-sharp corners. In anotherexemplary aspect, the base has rounded edges.

In another exemplary aspect, the cover has four rounded corners.

In another exemplary aspect, the device reduces damage to a membraneduring cryopreservation. In another exemplary aspect, the device issufficient to reduce damage to a membrane due to fluid shear forceduring addition or submersion into cryopreservation solution.

In another exemplary aspect, the device provides sufficient permeationof the cryopreservation solution to a membrane.

In another exemplary aspect, the device retains its integrity whenimmersed in a cryopreservation solution.

In another exemplary aspect, the device retains its integrity during afreeze-thaw cycle. In another exemplary aspect, the freeze thaw cycleincludes a freezing step of about −45° C. to −196° C. for a cellularmembrane and about −18° C. to about −196° C. for an acellular membrane.

In another exemplary aspect, the cover is the same size as the membranereceiving portion of the base.

In another exemplary aspect, the base and cover are free of impurities.In another exemplary aspect, the base and cover are free of particulatesor oils or other chemicals that may interfere with the viability ortherapeutic efficacy of the membrane.

In another exemplary aspect, the at least one location in which the baseand the cover are in communication comprises a plurality of points. Inanother exemplary aspect, the plurality of points comprise a pluralityof cauterization points, wherein the plurality of cauterization pointscomprises at least two cauterization points, preferably at least threecauterization points.

In another exemplary aspect, the at least one location in which the baseand cover are in communication allows for the retention of the cover tothe base through a cryopreservation step, a freezing step and a thawingstep.

In another exemplary aspect, a membrane is between the base and thecover. In another exemplary aspect, the membrane is attached to the baseby at least three points. In another exemplary aspect, the membrane isattached to the base by at least five points. In another exemplaryaspect, the membrane is attached to the base by at least six points. Inanother exemplary aspect, the cover is in communication with the base atleast at two points. In another exemplary aspect, the cover is incommunication with the base at least at three points. In anotherexemplary aspect, the membrane is a selected from the group consistingof a natural membrane, a synthetic membrane or a combination thereof. Inanother exemplary aspect, the membrane is a natural membrane. In anotherexemplary aspect, the membrane is a placental tissue product. In anotherexemplary aspect, the membrane is a synthetic membrane. In anotherexemplary aspect, the membrane is a combination of natural and syntheticmembrane. In another exemplary aspect, the membrane is bioengineered. Inanother exemplary aspect, the membrane is a chorionic membrane product.In another exemplary aspect, the membrane is an amniotic membraneproduct. In another exemplary aspect, the membrane is an in vitroderived tissue. In another exemplary aspect, the membrane is a culturedtissue equivalent.

In another exemplary aspect, the device withstands freezing at about−80° C. to about −196° C. without losing integrity.

In another exemplary aspect, the device is resistant to chemical orphysical alteration by cryopreservation solutions. In another exemplaryaspect, the cryopreservation solution comprises DMSO.

In another exemplary aspect, the membrane receiving portion is about 1.5cm×1.5 cm, about 2 cm×3 cm, about 3 cm×4 cm, or about 5 cm×5 cm.

In another exemplary aspect, the device allows for sufficient contactbetween the membrane and the cryopreservation solution to sufficientlycryopreserve the natural membrane to maintain viability. In anotherexemplary aspect, the viability is at least 70% after at least onefreeze-thaw cycle in cryopreservation solution. In another exemplaryaspect, the membrane maintains sufficient viability. In anotherexemplary aspect, the membrane has at least 70% viability.

In another exemplary aspect, the base and the membrane have sufficientsurface traction to maintain the membrane on the base.

In another exemplary aspect, the surface traction between the membraneand the cover is lower than the surface traction between the membraneand the base.

In another exemplary aspect, the plurality of perforations, plurality ofgrooves, or plurality of channels is sufficient to cryopreserve themembrane by providing contact between the membrane and thecryopreservation solution.

In another exemplary aspect, the membrane receiving portion of the baseis about 1.5 cm×1.5 cm, about 1.5 cm×2.0 cm, about 2 cm×2 cm, about 2cm×3 cm, about 3 cm×4 cm, about 5 cm×5 cm, about 5 cm×7 cm, or about 7.5cm×15 cm.

In another exemplary aspect, the handling portion comprises a label toindicate orientation. In another exemplary aspect, the label to indicateorientation is a word. In another exemplary aspect, the label toindicate orientation is a symbol.

In further exemplary aspects, the disclosed device can be used in amethod of maintaining the directionality of a membrane during storage,cryopreservation, or during application to a subject, the methodcomprising: a) preparing a tissue, wherein the tissue is orientatedhaving a first and a second side, wherein the first and second sidecomprise different composition; b) adhering the membrane to thedisclosed device, wherein the membrane is disposed between the base andthe cover, wherein the first side of the membrane is facing the base andthe second side of the membrane is facing the cover, and wherein thedevice further comprises a label to indicate orientation. In anotherexemplary aspect, the membrane is a placental tissue. In anotherexemplary aspect, the first side of the membrane comprises stromal cellsand the second side comprises epithelial cells. In another exemplaryaspect, the first side of the membrane has greater adherence to the basethan the second side of the membrane. In another exemplary aspect, thebase provides a handling portion that comprises the label to indicateorientation. In another exemplary aspect, the label is a word. Inanother exemplary aspect, the label is a symbol.

In still further exemplary aspects, the disclosed device can be used ina method of maintaining integrity of a membrane during cryopreservation,the method comprising: providing the disclosed device; adhering amembrane to at least an area of the membrane receiving portion of thebase; adhering the cover to the base, wherein the membrane is betweenthe cover and the base; and placing the device comprising the membraneinto a container; and contacting the container with sterilecryopreservation solution, wherein the device comprising the membrane issubmerged in the cryopreservation solution; and cryopreserving thecontainer at a temperature of about −80° C. to about −196° C. for anmembrane containing cells and about −18° C. to about −196° C. for anacellular membrane, wherein the integrity of the membrane is maintainedonce the membrane is thawed to room temperature.

In additional exemplary aspects, the disclosed device can be provided aspart of a kit, which further comprises instructions for adhering amembrane between the base and the cover of the device, wherein the baseand the cover have at least one location which is adapted to be incommunication with each other. In another exemplary aspect, the kitfurther comprises an adhesive that is biologically compatible. Inanother exemplary aspect, the instructions further comprise a method ofadhering the base to a first side of the membrane, wherein the methodcomprises applying the adhesive to at least one location between thebase and the membrane to form a membrane-covered base. In another Thekit of claim 156, wherein the instructions provide a method of adheringthe cover to base wherein the membrane is located between said cover andbase, wherein the method comprises applying the adhesive to at least onepoint between the cover and the membrane-covered base. In anotherexemplary aspect, the instructions further comprise a step ofcauterizing at least one point of the cover to the base, wherein themembrane is located between the cover and base. In another exemplaryaspect, the instructions further comprise a method of maintaining thedirectionality of the membrane, the method comprising the steps ofadhering a first side of the membrane to the base. In another exemplaryaspect, the kit further comprises instructions for cryopreserving thedevice comprising a membrane. In another exemplary aspect, the step ofcryopreserving comprises freezing the device containing the membrane at−80° C. In another exemplary aspect, the kit further comprisesinstructions for thawing the cryopreserved device. In another exemplaryaspect, the kit further comprises instructions of applying the membraneto a patient (human or animal) in need thereof.

Exemplary Compositions, Methods, and Kits

In further exemplary aspects, disclosed is a composition comprising: (a)a base comprising a membrane receiving portion; (b) a membrane; (c) acover; and (d) at least one location in which the base and the cover arein communication, wherein the membrane is positioned between the baseand the cover.

In another exemplary aspect, the base further comprises a handlingportion. In another exemplary aspect, the handling portion is adjacentto the membrane receiving portion. In another exemplary aspect, thehandling portion comprises a tab.

In another exemplary aspect, the membrane receiving portion comprises aplurality of perforations. In another exemplary aspect, the plurality ofperforations are uniformly distributed throughout the membrane receivingportion. In another exemplary aspect, the plurality of perforations arerandomly distributed throughout the membrane receiving portion. Inanother exemplary aspect, the plurality of perforations are evenlydistributed in close relationship to each other throughout the membranereceiving portion to provide sufficient surface traction for themembrane to adhere to the base. In another exemplary aspect, theplurality of perforations are about 1 mm to about 5 mm in diameter andthe each perforation is from 4 mm to about 10 mm apart as measuredcenter to center. In another exemplary aspect, the plurality ofperforations are from about 0.1 mm to about 5 mm diameter. In anotherexemplary aspect, the plurality of perforations are from about 0.1 mm toabout 1 mm diameter. In another exemplary aspect, the perforations arespaced about 0.35 mm to about 10 mm apart as measured center to center.In another exemplary aspect, the perforations are spaced about 4 mm toabout 10 mm apart as measured center to center. In another exemplaryaspect, the plurality of perforations are of any geometrical shape ornon-geometrical shape. In another exemplary aspect, the plurality ofperforations are a shape selected from the group consisting of oval,rectangular, square, diamond, trapezoid, star, hexagonal, octagonal,semi-circular, crescent, or a combination thereof. In another exemplaryaspect, the plurality of perforations are sufficient to cryopreserve themembrane by providing contact between the membrane and thecryopreservation solution.

In another exemplary aspect, the base and the cover comprise a singlesheet of plastic. In another exemplary aspect, the single sheet ofplastic is folded to form a base and a cover.

In another exemplary aspect, the membrane receiving portion is selectedfrom the group consisting of an abraded surface, a rough surface, ascratched surface, a surface comprising a plurality of perforations; asurface comprising a plurality of channels; a surface comprising aplurality of grooves; or a surface comprising a plurality ofindentations.

In another exemplary aspect, the base comprises at least one plastic.

In another exemplary aspect, the cover comprises at least one plastic.

In another exemplary aspect, the handling portion does not overlap withthe cover. In another exemplary aspect, the handling portion spans theentire width of the base. In another exemplary aspect, the cover spansthe entire membrane receiving portion of the base.

In another exemplary aspect, the base and the plastic cover are separatepieces of plastic.

In another exemplary aspect, the plastic of the base is selected fromthe group consisting of polycarbonate, copolyester, low densitypolyethylene (LDPE), high density polyethylene (HDPE), ECTFE copolymer,ETFE copolymer, FEP (fluorinated ethylene propylene), PE (Polyethylene),PP (Polypropylene), PMP (Polymethylpentene), Teflon, PS (Polystyrene),EVA, and Tritan copolyester MP100.

In another exemplary aspect, the plastic of the cover is selected fromthe group consisting of polycarbonate, copolyester, low densitypolyethylene (LDPE), high density polyethylene (HDPE), ECTFE copolymer,ETFE copolymer, FEP (fluorinated ethylene propylene), PE (Polyethylene),PP (Polypropylene), PMP (Polymethylpentene), Teflon, PS (Polystyrene),EVA, and Tritan copolyester MP100.

In another exemplary aspect, the cover is a solid sheet of plastic.

In another exemplary aspect, a first side of the cover is in contactwith a second side of the membrane and the first side of the membrane isin contact with a first side of the base, and wherein the first side ofthe cover has a lower surface tension than a first side of the base.

In another exemplary aspect, the cover has no sharp corners or edges. Inanother exemplary aspect, the corners are rounded.

In another exemplary aspect, the base has no sharp corners or edges. Inanother exemplary aspect, the base has rounded corners.

In another exemplary aspect, the composition is sufficient to reducedamage to the membrane during cryopreservation. In another exemplaryaspect, the composition is sufficient to reduce damage to the membranedue to fluid shear force during addition or submersion intocryopreservation solution.

In another exemplary aspect, the composition retains its integrity in acryopreservation solution.

In another exemplary aspect, the composition retains its integrityduring a freeze-thaw cycle. In another exemplary aspect, the freeze thawcycle includes a freezing step of −40° C. to −196° C. for a cellularmembrane and −18° C. to −196° C. for an acellular membrane.

In another exemplary aspect, the cover is the same size as the membranereceiving portion of the base.

In another exemplary aspect, the base and cover are free fromimpurities.

In another exemplary aspect, the base and the cover are free ofparticulates or oils or other chemicals that may interfere with theviability or therapeutic efficacy of the membrane.

In another exemplary aspect, the at least one location in which the baseand the cover are in communication comprises a plurality of points. Inanother exemplary aspect, the plurality of points comprise a pluralityof cauterization points.

In another exemplary aspect, the at least one location in which the baseand cover are in communication allows for the retention of the cover tothe base through a cryopreservation step, a freezing step and a thawingstep.

In another exemplary aspect, the membrane is attached to the base by atleast three points. In another exemplary aspect, the membrane isattached to the base by at least five points. In another exemplaryaspect, the membrane is attached to the base by at least six points.

In another exemplary aspect, the cover is in communication with the baseand membrane at least at two points. In another exemplary aspect, thecover is in communication with the base at least at three points.

In another exemplary aspect, the membrane is a selected from the groupconsisting of a natural membrane, a synthetic membrane or a combinationthereof.

In another exemplary aspect, the membrane is a natural membrane.

In another exemplary aspect, the membrane is a placental tissue product.In another exemplary aspect, the membrane is a chorionic membraneproduct. In another exemplary aspect, the membrane is an amnioticmembrane product.

In another exemplary aspect, the membrane is a synthetic membrane.

In another exemplary aspect, the membrane is a combination of naturaland synthetic membrane.

In another exemplary aspect, the membrane is bioengineered membrane.

In another exemplary aspect, the membrane is an in vitro derived tissue.

In another exemplary aspect, the membrane is a cultured tissueequivalent.

In another exemplary aspect, the membrane is a graft.

In another exemplary aspect, the composition withstands freezing at −80°C. without losing integrity.

In another exemplary aspect, the composition is resistant to chemicalalteration by a cryopreservation solution. In another exemplary aspect,the cryopreservation solution comprises DMSO.

In another exemplary aspect, the membrane receiving portion is 2 cm×2cm. In another exemplary aspect, the membrane receiving portion of thebase is about 1.5 cm×1.5 cm, about 1.5 cm×2 cm, about 2 cm×2 cm, about 2cm×3 cm, about 3 cm×4 cm, about 5 cm×5 cm, about 5 cm×7 cm, or about 7.5cm×15 cm.

In another exemplary aspect, the composition allows for sufficientcontact between the membrane and the cryopreservation solution tosufficiently cryopreserve the natural membrane to maintain viability.

In another exemplary aspect, the viability of the membrane is at least70% after at least one freeze-thaw cycle in cryopreservation solution.

In another exemplary aspect, the base and the membrane have sufficientsurface traction to maintain the membrane on the base when submerged ina solution.

In another exemplary aspect, the surface traction between a second sideof the membrane and the cover is lower than the surface traction betweena first side of the membrane and the base.

In another exemplary aspect, the membrane maintains sufficient viabilityafter cryopreservation. In another exemplary aspect, the viability ofthe membrane is at least 70%.

In another exemplary aspect, the membrane is a graft.

In another exemplary aspect, the base or the cover is labeled toindicate orientation. In another exemplary aspect, the label is locatedon the base. In another exemplary aspect, the label is located on ahandling portion of the base. In another exemplary aspect, the label islocated on the cover. In another exemplary aspect, the label is locatedon a handling portion adjacent to the cover.

In further exemplary aspects, the disclosed composition can be used in amethod of applying a membrane to a patient (human or animal) in needthereof, the method comprising: obtaining the disclosed composition,wherein the composition has been cryopreserved and frozen; thawing thecomposition; rinsing the membrane in a sterile physiological solution;removing the cover from the membrane and base; and applying the membranefrom the base onto the patient (human or animal) to retaindirectionality of the membrane.

In still further exemplary aspects, the disclosed composition can beused in a method of treating a wound, the method comprising applying amembrane of the composition to a patient in need thereof.

In additional exemplary aspects, the disclosed composition can beprovided as a cryopreserved membrane composition, which furthercomprises a cryopreservation solution. In another exemplary aspect, thecryopreservation solution comprises DMSO. In another exemplary aspect,the cryopreservation solution comprises about 5% DMSO. In anotherexemplary aspect, the cryopreservation solution comprises about 2% toabout 10% DMSO. In another exemplary aspect, the cryopreservationsolution further comprises about 1% to about 20% serum albumin. Inanother exemplary aspect, the cryopreserved membrane composition furthercomprises physiological saline.

In further exemplary aspects, the disclosed cryopreserved membranecomposition can be provided as part of a kit, which further comprisesinstructions for applying the cryopreserved membrane to a tissue defect.In another exemplary aspect, the kit further comprises instructions forthawing the cryopreserved composition. In another exemplary aspect, thekit comprises further instructions on maintaining the directionality ofthe membrane while being applied to the tissue defect. In anotherexemplary aspect, the tissue defect is a wound. In another exemplaryaspect, the kit further comprises instructions for removal of the cover.In another exemplary aspect, the kit further comprises instructions formaintaining the directionality of the membrane. In another exemplaryaspect, the kit further comprises instructions for removing the membranefrom the base.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

What is claimed is:
 1. A membrane product package comprising: a basehaving a longitudinal axis and comprising a product receiving portion,the product receiving portion having a top surface and an opposed bottomsurface that are spaced apart relative to a vertical axis that isperpendicular to the longitudinal axis of the base, wherein the productreceiving portion comprises a traction-creating feature, wherein thetraction-creating feature comprises one or more of the following: (i) arough top surface, (ii) a plurality of perforations extending betweenthe top and bottom surfaces of the product receiving portion, (iii) asurface comprising a plurality of channels, (iv) a surface comprising aplurality of grooves, (v) a surface comprising a plurality ofindentations, or (vi) a surface comprising a plurality of porations; anda cover having a longitudinal axis, a top surface, and an opposed bottomsurface, wherein the cover is configured for releasable coupling to thebase in a product-covering position, and wherein, in theproduct-covering position, the cover overlies the product receivingportion of the base; and a membrane positioned in an operative positionbetween the product receiving portion of the base and the cover, whereinthe membrane is a placental tissue product, a tissue graft, an in vitrocultured graft, a tendon graft, or a bioengineered membrane comprisingliving cells, wherein the membrane is positioned in engagement with atleast a portion of the top surface of the product receiving portion ofthe base and at least a portion of the bottom surface of the cover,wherein the base and the cover cooperate to support the membrane in theoperative position.
 2. The membrane product package of claim 1, whereinthe base further comprises a handling portion position adjacent to theproduct receiving portion relative to the longitudinal axis of the base.3. The membrane product package of claim 2, wherein, in theproduct-covering position, the cover does not overlap with the handlingportion of the base.
 4. The membrane product package of claim 1, whereinin the product-covering position, the longitudinal axis of the cover ispositioned in a substantial alignment with the longitudinal axis of thebase.
 5. The membrane product package of claim 4, wherein the productreceiving portion of the base has a longitudinal length and a width,wherein the cover has a longitudinal length and a width, and wherein thelongitudinal length of the cover 1 s substantially equal to thelongitudinal length of the product receiving portion.
 6. The membraneproduct package of claim 5, wherein the width of the cover issubstantially equal to the width of the product receiving portion. 7.The membrane product package of claim 1, wherein the cover has aplurality of comers, and wherein at least one of the comers of the coveris rounded.
 8. The membrane product package of claim 7, wherein thecover has four rounded comers.
 9. The membrane product package of claim8, wherein the product receiving portion of the base has two roundedcomers, and wherein, in the product-covering position, two roundedcomers of the cover overlie the two rounded comers of the productreceiving portion of the base.
 10. The membrane product package of claim1, wherein the traction-creating feature of the product receivingportion comprises a plurality of perforations.
 11. The membrane productpackage of claim 10, wherein the plurality of perforations of theproduct receiving portion of the base are substantially evenlydistributed throughout the product receiving portion.
 12. The membraneproduct package of claim 10, wherein the plurality of perforations ofthe product receiving portion of the base are randomly distributedthroughout the product receiving portion.
 13. The membrane productpackage of claim 10, wherein each perforation of the plurality ofperforations has a respective diameter ranging from about 0.1 mm toabout 5 mm.
 14. The membrane product package of claim 10, wherein eachperforation of the plurality of perforations has a respective centerpoint, and wherein the center points of neighboring perforations arespaced apart by a distance ranging from about 0.35 mm to about 10 mm.15. The membrane product package of claim 1, wherein the membrane isattached to the top surface of the product receiving portion of the baseat least one attachment point.
 16. The membrane product package of claim15, wherein the membrane is attached to the top surface of the productreceiving portion of the base at least three attachment points.
 17. Themembrane product package of claim 15, wherein the top surface of theproduct receiving portion of the base is attached to the cover at leastone attachment point.
 18. The membrane product package of claim 15,wherein the cover is attached to the membrane at least one attachmentpoint.
 19. The membrane product package of claim 18, wherein the coveris attached to the membrane at least two attachment points.
 20. Themembrane product package of claim 18, wherein the top surface of theproduct receiving portion of the base is attached to the cover at leastone attachment point.
 21. The membrane product package of claim 1,wherein the top surface of the product receiving portion of the base isattached to the cover at least one attachment point.
 22. The membraneproduct package of claim 1, wherein the membrane is a chorionic membraneproduct.
 23. The membrane product package of claim 1, wherein themembrane is an amniotic membrane product.
 24. The membrane productpackage of claim 1, wherein the membrane and the top surface of theproduct receiving portion of the base have sufficient surface tractionto maintain the membrane in the operative position following removal ofthe cover from the base.
 25. The membrane product package of claim 1,wherein the membrane and the top surface of the product receivingportion of the base have a first surface traction, wherein the membraneand the cover have a second surface traction, and wherein the secondsurface traction is lower than the first surface traction.
 26. Themembrane product package of claim 1, wherein the traction-creatingfeature of the product receiving portion comprises a rough top surface.27. A method of producing a membrane product package, comprising:positioning a membrane in an operative position between a productreceiving portion of a base and a cover, wherein the membrane ispositioned in engagement with at least a portion of a top surface of theproduct receiving portion of the base and at least a portion of a bottomsurface of the cover, and wherein the membrane is a placental tissueproduct, a tissue graft, an in vitro cultured graft, a tendon graft, ora bioengineered membrane comprising living cells, and wherein theproduct receiving portion of the base comprises a traction-creatingfeature, and wherein the traction-creating feature comprises one or moreof the following: (i) a rough top surface, (ii) a plurality ofperforations extending between the top and bottom surfaces of theproduct receiving portion, (iii) a surface comprising a plurality ofchannels, (iv) a surface comprising a plurality of grooves, (v) asurface comprising a plurality of indentations, or (vi) a surfacecomprising a plurality of porations.
 28. The method of claim 27, whereinthe step of positioning the membrane in the operative positioncomprises: attaching the membrane to the top surface of the productreceiving portion at least one attachment point; and attaching themembrane to the cover at least one attachment point.
 29. The method ofclaim 28, wherein the traction-creating feature of the product receivingportion of the base comprises a plurality of perforations, wherein themethod further comprises positioning the base, the membrane, and thecover within a cryopreservation solution, wherein the plurality ofperforations of the product receiving portion provide contact betweenthe membrane and the cryopreservation solution sufficient tocryopreserve the membrane.
 30. A kit for repairing a tissue defect,comprising: a container; and a membrane product package positionedwithin the container, the membrane product package comprising: (a) asupport assembly having: (i) a base having a longitudinal axis andcomprising a product receiving portion, the product receiving portionhaving a top surface and an opposed bottom surface that are spaced apartrelative to a vertical axis that is perpendicular to the longitudinalaxis of the base, wherein the product receiving portion comprises atraction-creating feature, wherein the traction creating feature isselected from the group consisting of (i) a rough top surface, (ii) aplurality of plurality of perforations extending between the top andbottom surfaces of the product receiving portion, (iii) a surfacecomprising a plurality of channels, (iv) a surface comprising aplurality of grooves, (v) a surface comprising a plurality ofindentations, or (vi) a surface comprising a plurality of porations; and(ii) a cover having a longitudinal axis, a top surface, and an opposedbottom surface, wherein the cover is configured for releasable couplingto the base in a product-covering position, and wherein, in theproduct-covering position, the cover overlies the product receivingportion of the base; and (b) a membrane positioned in an operativeposition between the product receiving portion of the base and thecover, wherein the membrane is positioned in engagement with at least aportion of the top surface of the product receiving portion of the baseand at least a portion of the bottom surface of the cover, and whereinthe membrane is a placental tissue product, a tissue graft, an in vitrocultured graft, a tendon graft, or a bioengineered membrane comprisingliving cells, wherein the base and the cover cooperate to support themembrane in the operative position.
 31. The kit of claim 30, furthercomprising a cryopreservation solution.